DE10309803A1 - New alpha-amylase variants, useful in cleaning and washing compositions and for desizing cotton, have improved activity in alkaline media, also nucleic acid that encodes them - Google Patents

Abstract

Variants (A) of alpha -amylase having at least two amino acids (aa) exchanged, relative to a starting molecule,and including Pro at position 13 and Ser at position 414 of the alpha -amylase sequence of Bacillus amyloliquefaciens (sequence (4) of 514 aa) are new. Independent claims are also included for the following: (1) similar variants (A1) with exchanges at positions 203 and 474 of sequence (4); (2) nucleic acid (I) that encodes (A) or (A1); (3) vector that contains (I); (4) cell that contains (I), especially as the vector of (3); (5) method for preparing (A) or (A1); and (6) composition (B) that contains (A) or (A1).

Description

The present invention relates to α-amylase variants with an improved activity in an alkaline medium and various technical fields of application for these α-amylase variants, in particular Detergents and cleaning agents with these α-amylase variants.

α-amylases (E.C. 3.2.1.1) hydrolyze internal α-1,4-glycosidic bonds of strength and starch-like Polymers to form dextrins and β-1,6-branched oligosaccharides. by virtue of this divisive starch activity become α-amylases for numerous technical applications, including as an active component in detergents and cleaning agents. Because detergents and cleaning agents are predominant have alkaline pH values, especially α-amylases needed that are active in the alkaline medium. Most of the technically important α-amylases are from Microorganisms, i.e. Fungi or bacteria, especially those of the Genera Aspergillus and Bacillus produced and secreted. outgoing from these microbiological sources, they can mostly be (genetically) technical adjusted accordingly Industrial scale processes be won.

To determine suitable α-amylases for the respective areas, an intensive search for new α-amylases from natural sources continues to take place. Many such enzymes have already been described in the prior art; their number continues to increase. The alkaline α-amylases, which are naturally formed by species of the genus Bacillus, are of particular importance for use at alkaline pH values. The α-amylases from Bacillus licheniformis, from B. amyloliquefaciens and from B. stearothermophilus are of particular technical importance. In the present application they are referred to 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 α-amylase suitable for use in alkaline media (referred to in the present application as LAMY) is described by 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). Also to be mentioned is the α-amylase S707, originally from Bacillus sp. # 707 has been isolated 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. In Biochem. Biophys. Res., Comm. (1988), 151 (1), 25-31.

More examples of special amylolytic enzymes that can be used in detergents and cleaning agents the α-amylase from Bacillus sp. A 7-7 (DSM 12368; referred to here as A 7-7) in WO 02/10356 A2, and the enzyme from B. agaradherens (DSM 9948), that at the same time as α-amylase and be viewed as cyclodextrin glucanotransferase (CGTase) can and has been published in WO 02/44350 A2. Other α-amylases are those in the sequence space defined in the application WO 03/002711 A2 belong.

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

Another Bacillus amylase is 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 referred to here as TS-23. The associated DNA and amino acid sequences are, for example, in the 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) filed under number U22045.

A comparison of the sequences of these enzymes requires an alignment in which the amino acids corresponding to one another come to lie on top of one another. The amino acid positions relating to the present invention, discussed in detail below, can be derived from the alignment of the following with these most important enzymes 1 derived table can be assigned. The numbering of the α-amylase from B. amyloliquefaciens (BAA) given in the first line is that according to SEQ ID NO. 4th

Table 1: Allocation of amino acid exchanges 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 amino acids are international geräuchlichen Single letter code specified; Abbreviations of the Enzymes as in the text; K. A. stands for: no information.

The naturally occurring α-amylase molecules are in many ways, especially through molecular biological modifications have been developed to meet specific applications to optimize. Such optimizations concern, for example the substrate specificities, the stability of the enzyme under different conditions or the enzymatic activity self.

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

However, this is still an important one The principle of making such further developments is the implementation of Point mutations, i.e. Deletions, insertions and above all substitutions single amino acids. For example, the random mutagenesis process 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 meanwhile numerous variants improved by point mutations have been described, these variations usually not the enzymes in question are restricted 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 and thereby to reduce the sensitivity to oxidation and to improve the activity at higher temperatures. This is illustrated using variants of the α-amylase from B.licheniformis. Further improvements to this Termamyl ^® molecule go for example from WO 95/26397 A1 forth. Such optimization products of the α-amylase from B. licheniformis are available under the trade name Duramyl ^® by the company. Novozymes (SOFW-Journal, 123, (1997), pp 723-731). Another optimization product is Termamyl ^® ultra with less calcium binding.

The α-amylases of application WO 97/43424 A1 show about Point mutations in the calcium-binding amino acids change the calcium ion binding behavior and thus changed enzymatic properties. This knowledge is based on the 3D structure of α-amylase was obtained from B. licheniformis, but was also on other α-amylases applicable. The application WO 96/23874 A1 is also based on knowledge from the 3D structure of different molecules, including also hybrid amylases, and strikes numerous positions at which α-amylases, especially the "classic" molecules BLA, BStA or BAA changed can be to change their enzymatic properties.

For example, from 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 less calcium dependence. The amylases of application WO 99/02702 A1 with additional disul fid bridges are more stable than the parent molecule at higher temperatures. 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. Derived variants, in particular of the α-amylase from B. stearothermophilus, can be found, for example, in application WO 02/02726 A1 for use in detergents and cleaning agents. The corresponding commercial product from Novozymes bears the name Novamyl ^® .

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

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

Like the wild type enzymes, too Hybrid amylases additionally be further developed by point mutagenesis. This reveals, 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 that are modified in terms of Cleavage pattern on the starch substrate have been developed and are therefore particularly suitable for starch processing suitable. In these writings there are numerous point mutations both of native α-amylases, as well as hybrid amylases.

Compared to the ones presented here Documents from the prior art are the following the present Positions and substitutions relevant to registration are checked.

From the application WO 97/41213 A1 Variants emerge, which in the BLA count the exchanges D325N, R375E in combination with another substitution and L3V, in combination with several other defined exchanges exhibit.

The application WO 00/60059 A2 discloses a variety of ways to use α-amylases by changing point mutations. Among them, an exchange in position S168 (according to BLA) is proposed, specifically the variant S168Y.

From the registration EP 985731 A1 there are α-amylase variants which can be obtained by mutation in position A202 according to LAMY; in particular by exchanging for T, I, L, A, V or S. Of these, the SEQ ID NO. 4 affected position A230 relevant to the invention.

From the application WO 96/23874 A1, numerous variants which influence the enzymatic activity emerge, which have been suggested by comparing the 3D structures of different α-amylases. However, this does not include an exchange in a position relating to the present application. The corresponding patent US 5989169 also describes a method for recognizing those positions which relate to the calcium dependency or the stability of the relevant enzymes by comparing the 3D structures of different α-amylases. Only in this special context are the amino acid positions A199 and Q443 of BLA mentioned in addition to numerous other positions. A pre-description of the fact that these positions could possibly relate to the alkali activity is not to be seen in it.

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

The application WO 95/10603 A1 discloses again a variety of substitutions that include the contributions of those concerned Enzymes to affect washing performance, specified in the count BLA. This should include exchanges for the general areas only are specified; from areas 142–182, 172–178, 260–269 and 369-383 only position 375 is specifically described. For this the Exchange R375Y revealed. Especially in the latter area position 383 is given as a limit without this Position, however, is given meaning, so that probably only the amino acids between the edge positions appear to be meant. At least no example reveals that it is precisely this position that unites Influence on the alkali activity takes. Another position of relevance to this document is position 3 out; is recommended for this the exchange L3V.

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

So none of this registration Substitutions in the amino acid positions 174 and 383, corresponding to 203 and 414 in the counting according to SEQ ID NO. 4 out. On the other hand it means it only in WO 02/092797 A2 that these variants had been created; however, information on the biochemical properties is missing. For none However, these positions will have an impact on usability described alkaline conditions.

In the above application WO 99/23211 A1 are also called variants of the enzymes SP690 and SP722, which concern positions W167, S170, D271 and K385, whereby, according to Claim, each substituted by all remaining amino acids shall be; which means in general that here in principle every amino acid possible is. Only the K385R exchange is specifically disclosed, i.e. the substitution of one basic amino acid for another basic amino acid at this position, in combination with other point mutations.

In summary, it should be noted that no exchanges in positions 13 and 203 according to the counting according to SEQ ID NO. 4 are described above. In position 203, as for example from Table 1 and from the alignment of the 1 an isoleucine residue (I) is highly conserved in most α-amylases. Position 13 is within the propeptide. For position 414, only the replacement of a basic amino acid by another basic amino acid has actually been disclosed, which correlates with the fact that in most other α-amylases the amino acid lysine (K) is located here. A substitution against another amino acid, especially against the amino acid serine, has not yet been described. In each case, some of the other positions considered in the present invention have already been varied in the prior art, albeit only rarely with regard to the new amino acids specifically proposed here and never in these position combinations.

From this introductory presentation it is also clear that a unbroken need thereafter, there are new ones and especially with regard to various possible uses improved α-amylases to find in nature or to develop in a targeted manner. In particular for the Use in detergents and cleaning agents results in the need for further development from the various cleaning purposes changing habits and claims of the consumer, for example with regard to lower cleaning temperatures or special dosage forms or from further development other ingredients that make the enzymes perform affect, such as bleaching compounds. But especially have the most commercially available Detergents and cleaning agents have a strongly alkaline pH value, so that one Improve activity enzymes in question at high pH values a permanent challenge represents the optimization of detergent and cleaning agent enzymes.

So the task was new α-amylases or α-amylase variants to develop that show better conditions to work within the framework to develop the highest possible activity of detergent and cleaning agent formulations and thus to improve their washing or cleaning performance. Which also includes especially improved activity under alkaline conditions.

A subtask existed with regard on the industrial applicability of this development in that for such α-amylases coding nucleic acids to disposal to provide the relevant enzymes molecular biological developments and to make technical manufacturing processes accessible.

The object is achieved according to the invention by providing α-amylase variants with at least two amino acid exchanges with respect to the starting molecule, which are characterized in that in position 13 according to the counting of the α-amylase from B. amyloliquefaciens (SEQ ID NO. 4) a proline residue (P) and in position 414 a serine residue (S) is present. According to the invention, another aspect of achieving the object is provided by providing α-amylase variants with at least two amino acid exchanges with respect to the starting molecule, which are characterized in that an exchange in position 203 and an exchange at position 474 according to the B. amyloliquefaciens α-amylase (SEQ ID NO. 4) is present. Both aspects are illustrated by corresponding variants, which are described in the examples for the present application and represent corresponding preferred embodiments of the present application (see below).

According to the invention and as explained in the introduction, α-amylases are to be understood as meaning enzymes of the classification EC 3.2.1.1 which hydrolyze internal α-1,4-glycosidic bonds of starch and starch-like polymers with the formation of dextrins and β-1,6-branched oligosaccharides. In principle, the invention is applicable to all known and still to be found α-amylases, insofar as they can be homologized with the α-amylase from B. licheniformis. Such a homologation is in 1 for the technically most important α-amylases and it allows or facilitates finding the amino acid positions corresponding to the invention in the other amylases. As explained further below, the invention is preferably 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 α-amylases established in the prior art are given in the introduction.

According to the invention, variants are to be understood as meaning proteins which differ from starting proteins with regard to the amino acid sequence. The counterpart to this at the nucleic acid level is the mutant. For example, BAA L13P is a variant of the BAA that has all amino acids except the one in position 13 in common with the BAA. At the DNA level, this corresponds to the exchange of the codon CTT (37–39 according to SEQ ID NO. 3) against CCT (according to SEQ ID NO. 5).

Amino acid exchanges are to be understood as substitutions of one amino acid for another amino acid. According to the invention, such substitutions are specified in the internationally customary single-letter code, indicating the positions in which the exchange takes place, optionally combined with the amino acids in question. "Exchange in position 13" means, for example, that a variant in the position which has the 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 denotes a sequence difference such exchanges are carried out at the DNA level via mutations of individual base pairs. “L13P” means, for example, that the reference enzyme at position 13 has the amino acid leucine, while the variant under consideration has the amino acid proline at the position that can be homologated with it. "13P" means that any, that is to say a naturally predetermined amino acid, is replaced by a proline at a position which corresponds to position 13. An example of such a homologous exchange would be L-18P in BLA, ie the α-amylase formed by B.licheniformis, because over 1 have position 13 of BAA homologated with position -18 of BLA.

Basically, those with the present Registration does not suggest proposed amino acid exchanges limited to that she the only exchanges are in which the variant in question of the wild type molecule different. It is known in the prior art that the advantageous properties of individual point mutations can complement each other. A with regard to certain properties such as calcium binding or stability across from Α-Amylase optimized for surfactants can additionally according to the invention Substitutions presented here are further developed. Consequently include embodiments of the present invention all variants that interchange among others across from the wild type molecule also the exchanges according to the invention exhibit. This is especially true for molecules that have a fusion mutation (hybrid formation) have been obtained (see below).

According to the invention, the unprocessed 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 lines in the alignments of the 1 and 2 (BAA). The variants according to the invention are described using 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 a so-called propetide. For some enzymes such as AA349 and AA560, only the mature sequence parts are given in the patent literature (see above). Homologation can only be carried out via the relevant positions of the mature area and is corresponding by sequence comparison 1 possible.

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

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

In position 414, most other α-amylases have a basic amino acid (cf. Tables 1 and 1 ). Accordingly, only the replacement of a basic amino acid with another basic amino acid has been suggested by the prior art in this position so far. It is therefore 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 Registration is for three variants of the α-amylase from B. licheniformis (BAA A42, BAA A29 and BAA B1) in detail described. You have across from this comparison molecule (the Wild type enzyme), measured at pH 6.5 above significantly higher specific activities (see example 5). additionally opposes the variant BAA A42 the wild type molecule one shifted by an entire pH unit into the alkaline range pH optimum. Also has them over a significantly increased relative activity at pH 10 (see example 6).

These three variants have two things in common, namely the two amino acid exchanges L13P and N414S. Thus it can be assumed according to the invention that this both exchanges the activity of α-amylases, especially their alkali activity able to increase.

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

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

The success of these exchanges is demonstrated by the BAA variant A42, whose DNA and amino acid sequences are under SEQ ID NO. 5 and 6 are specified. Example 5 shows the increase in performance and example 6 shows the particularly high performance in an alkaline medium.

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

It is particularly preferred a variant with the exchanges 13P / 194R / 197P / 356D / 414S.

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

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

Position 32 is the first amino acid of the mature protein in BAA, and is the third amino acid of the mature protein in most other α-amylases. It may play a certain role in the folding of the protein. Position 230 (242 according to 1 ) is naturally occupied by an alanine residue in most other α-amylases and is in a highly conserved range. It is therefore surprising that a successful exchange can take place in such a position. The latter also applies to position 297 (309 according to 1 ), which in the BAA is naturally occupied by an N, i.e. an amino acid, with an acid amide side chain. In position 406 (418 according to 1 ) there is naturally a K at the BAA; the most important α-amylases from the prior art have the amino acid residues R, Y or H there, so they tend to show a mixed picture; this is particularly because Y has a hydroxyl group.

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

In particular for position 230 was not closed expected to be here a more space-filling amino acid successfully introduced as alanine can be. Is surprising also that the better suitability of the variants according to the invention for alkaline Conditions apparently only to a small extent 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 that at position 406 from a basic to a stronger one basic amino acid.

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

Such a molecule is used with the BAA variant A29 available put and in the examples regarding its biochemical Properties examined. It shows particularly high activity values compared to the wild type enzyme, i.e. the molecule without these six exchanges on.

As already mentioned above, there is a another aspect to the solution the task in the provision of α-amylase variants with at least exchange two amino acids across from the starting molecule, which are characterized in that an exchange in position 203 and an exchange at position 474 according to the B. amyloliquefaciens α-amylase count (SEQ ID NO. 4).

This can be exchanges act on a wild-type molecule, or in particular to those that are connected to an existing and possibly made in a certain direction improved variant additionally have been.

Position 203 is how 1 shows in a relatively conserved range of α-amylases; isoleucine in particular is present at this point in all other important α-amylases. The position looks more indifferent for position 474, because here there are various amino acids in the comparative enzymes from the prior art, in particular K, H, N or Q. Nevertheless, there is nothing to suggest that these positions are important for alkali stability and in particular the alkali activity could be important.

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

Because about these two substitutions the variant BAA B1 according to the invention differs from BAA A29. This gives it, as Example 6 shows, with a non-measurably changed pH optimum a higher relative activity at pH 10.

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

Such α-amylase variants are thus claimed, the additional to the exchanges mentioned in positions 13 and 414, in combination marked with 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 the α-amylase from B. amyloliquefaciens (SEQ ID NO. 4) is present, preferably in both positions.

According to what is said above also among these α-amylase variants preferred those characterized in that in position 203 a leucine (L) and / or in position 474 a glutamine residue (Q) is present.

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 deals with the exchanges 13P / 32A / 203L / 230V / 297D / 406R / 414S / 474Q.

A representative of such a molecule is obtained according to Examples 1 to 3 and in Examples 4 to 6 characterized variant BAA B1, which also has the name B. amyloliquefaciens-α-amylase L13P / V32A / 1203L / A230V / N297S / K406R / N414S / K474Q wearing. It stands out the wild type molecule BAA in particular through an increased activity from (Example 5).

As introduced in the introduction State of the art numerous α-amylases for the Use for various technical purposes, especially as an active one Components established in detergents and cleaning agents. These include fungal ones and especially bacterial enzymes. Very common are those from gram-positive bacteria, especially of the genus Bacillus adapted to an alkaline environment. Because they point from cheap in advance Properties for these areas of application. According to the invention, they can be added Point mutations are further developed. In the scope of protection this application falls thus generally every α-amylase variant, which are characterized by the amino acid variations indicated above is, preferably each, for a corresponding use has already been described, for example also the molecules AA560, AA349, SP690 and SP722.

This is preferably any α-amylase variant, which is characterized in that the starting molecule is an α-amylase derives that originally comes 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 E.g. A 7-7 (DSM 12368), very particularly from B. amyloliquefaciens.

These enzymes have been dealt with in detail in the introduction. Particularly preferred are those which derive from the α-amylases from B. agaradherens (DSM 9948) and from B. sp. Derive A 7-7 (DSM 12368). The first-mentioned molecule, which can be regarded as α-amylase and cyclodextrin glucanotransferase (CGTase) at the same time, is described in application WO 02/44350 A2 wrote. The α-amylase from Bacillus sp. A 7-7 (DSM 12368), which is referred to here as A 7-7, emerges from the application WO 02/10356 A2. No improvements in performance via point mutations have yet been described for either, so that the variations according to the invention are likely to be particularly promising first starting points for this. On the other hand, variants of the enzyme from B. amyloliquefaciens (BAA) are preferred because they have been produced according to the examples given here and have the advantageous properties mentioned.

As mentioned, the present invention not on natural or largely unmodified enzymes but can in particular can also be applied to hybrid amylases. Because the production of α-amylases with positive characteristics about fusion mutagenesis has now been described in detail in the prior art.

Accordingly, every α-amylase variant according to the invention claimed that additionally is characterized in that it itself with the starting molecule a hybrid α-amylase acts, 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), particularly 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).

Especially from the registration DE 10138753 hybrids of α-amylases from B. amyloliquefaciens and B. licheniformis emerge, which show positive properties especially when used in detergents and cleaning agents.

Each is therefore particularly preferred α-amylase variant according to the invention, which is characterized in that it is in the hybrid α-amylase is one whose partial sequences from the α-amylases from B. amyloliquefaciens and are derived from B. licheniformis.

An α-amylase variant is particularly preferred, if it is characterized by the fact that it is a variant the α-amylase B. amyloliquefaciens with one of the amino acid exchange combinations L13P / W194R / S197P / E356D / N414S, L13P / V32A / A230V / N297D / K406R / N414S or L13P / V32A / 1203UA230V / N297D / K406R / N414S / K474Q deals, preferably with the amino acid exchange combination L13P / W194R / S197P / E356D / N414S.

Because these molecules are under the names BAA 42, BAA 29 and BAA B1 in the examples of the present application described. Below that is the enzyme with the amino acid exchange combination L13P / W194R / S197P / E356D / N414S (BAA A42) have a particularly high alkaline activity Provided proof.

These are preferably α-amylase variants characterized in that it 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 fact, as in example 1, the molecules BAA A42, A29 and B1 not from strains of the species B. amyloliquefaciens itself. Originally came from just 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 1b, 38124 Braunschweig (http://www.dsmz.de) can be obtained. This is under SEQ ID NO.3 respectively 4 specified. The relevant mutant B. amyloliquefaciens strains can, for example are generated in that the shown pGBAA plasmids preferably derived therefrom plasmids with larger flanking Areas of the BAA gene are transferred back into the wild-type cells. They could be analog Genes from SEQ ID NO. 5, 7 and 9 also newly synthesized and in corresponding Vectors are introduced. By homologous recombination of the mutated Gens in the associated The demanding mutant of B. amyloliquefaciens is located in the chromosome generated that 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.

Your own subject of the invention represent the nucleic acids that for one of the α-amylase variants described so far encode.

These include both RNA and DNA molecules as well as understanding DNA analogs, both coding and codogenic strand, in every reading frame. Because, for example would it be possible that to use the teaching associated with the invention to a interfering corresponding RNA regulation of corresponding α-amylases or increase the lifespan of the genetic information by it is converted into a slower degradable DNA analog in vivo. The nucleic acids open, like the ones detailed below Invention objects can be seen, the present invention, so to speak molecular biological dimension. They are in accordance with the above accordingly preferred.

These are preferably to be understood as DNA molecules. Because about this is it possible the α-amylase variants according to the invention per se to produce known molecular biological methods and / or if necessary weiterzumodifizieren.

Starting points for this nucleic acid can be the DNA Se specified in the sequence listing be sequence information in order to obtain the relevant genes via chemical resynthesis. In contrast, it can also be advantageous to use nucleic acids that are already available in vivo, for example those that code for the α-amylases known in the prior art. Another alternative is to carry out a total DNA preparation of certain microorganisms envisaged therefor in accordance with Example 1 and to isolate the endogenous α-amylase genes by means of PCR. In principle, those from SEQ ID NO. 3 5 'and 3' sequence sections, which can also be read, are used.

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

This does not only mean those which as explained above have been obtained from this sequence, but also all mutants, that have been created in positions other than just those that are described with the present application.

A preferred embodiment accesses those actually examined in the present application Examples back.

Each is therefore particularly preferred nucleic acid according to the invention, the is characterized in that it different from a nucleic acid according to SEQ ID NO. 5, SEQ ID NO. 7 or SEQ ID NO. 9 derives, preferably one of these sequences themselves.

A particularly advantageous form nucleic acids to handle are vectors. Because these have areas that allow to characterize the contained gene of interest (about about flanking areas for Segregation primer), and if necessary by transformation reactivate it or to express it if the derived one Protein examined and produced in large quantities shall be. For vectors, it can be especially for eukaryotic ones Host cells such as yeast around artificial Act chromosomes; in bacteria, vectors are usually plasmids.

Your own subject of the invention vectors for implementing the present invention which contain a region with a nucleic acid described above.

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

Because in this form the concerned DNA can be stored as mentioned above or, for example, by introducing additional ones Mutations further changed are, for example, further developed α-amylase variants according to the invention can be generated.

In another preferred embodiment such a vector is characterized in that it is an expression vector is.

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

In further advantageous embodiments the vectors mentioned indicate origins of replication for different microorganisms on, which makes them so-called shuttle vectors into different Host cells can be introduced and stably maintained there. It is also possible To produce vectors, both as cloning and as Expression vector can be viewed.

According to the above and The methodology established in the prior art extends the molecular biological dimension also on organisms, in particular on microorganisms.

Your own subject of the invention thus represent cells which are characterized in that they have a of the nucleic acids described above included, preferably on a vector referred to above.

These include, for example, which have been transformed with an appropriate cloning vector are or are derived from such. This serves in particular the Characterization and / or storage of the gene in question. In contrast is it for the realization of the present invention is particularly important actual enzyme in question also get; because its improved properties exist a particular commercial interest. This education can for example permanent and in high production rate if that is of interest Transgene under the control of a constitutive and strong promoter stands. Alternatively, it is also possible the gene for the α-amylase variant under the control of an inducible and advantageously not less powerful promoter. This allows the manufacture targeted from the outside be switched on, for example, if there are favorable conditions for this. This is the case, for example, with fermentative production if a certain cell density has been reached.

So each is preferred above Cell which is characterized in that it is one of the α-amylase variants mentioned above forms or can be stimulated to form them, preferably using a nucleic acid described above, particularly preferably under Use of an expression vector described above.

An additional advantageous embodiment refers to the fact that most industrially important α-amylases were originally found as enzymes which are secreted into the surrounding medium by the microorganisms in question, in particular bacteria. Because in vivo it is mostly about Digestive enzymes. Accordingly, it is advantageous if the industrially produced enzymes according to the invention are also secreted into the surrounding medium. Because from this they can be processed without cell disruption and thus comparatively easily. This can be achieved, for example, by adding appropriate sequences to the genes in question - if these are not already present - which code for a leader peptide which causes the cell in question to release them. An alternative in the case of Gram-negative bacteria is, for example, to partially open the outer membrane for the release of proteins, as is described, for example, in the application WO 01/81597 A1.

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

In particular the characterization genes and enzymes are usually produced on a laboratory scale by intermediate cloning and / or expression in gram-negative bacteria. This is also proven the examples of the present application. But they are also production processes established that build on gram-negative bacteria (see above).

The cells according to the invention described so far are therefore preferably characterized in that it is Gram-negative bacteria, preferably of the genera Escherichia coli or Klebsiella, especially derivatives of E. coli K12, from E. coli B or K. planticola, and especially around derivatives of the tribes E. coli BL21 (DE3), E. coli RV308, E. coli DH5α, E. coli JM109, E. coli XL-1 or K. planticola (RF).

The prior art is particularly for the Production of enzymes from gram-positive organisms established for this also choose gram-positive expression hosts. But also regarding the characterizations on a laboratory scale, it is not uncommon to use gram positive bacteria to work. It is particularly advantageous if a particular enzyme is also produced by the host where it originally came from because this regulating signals generally optimal be implemented.

The cells according to the invention described above are therefore no less preferably characterized in that it is these are gram-positive bacteria, especially of the genera Staphylococcus, Corynebacterium or Bacillus, especially 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 finally, still all methods for producing an α-amylase variant according to the invention Act. Because this makes the object that is actually of economic interest indeed to disposal posed. Such a production can be done, for example, via a chemical synthesis of the protein in question.

In contrast, are according to the known and molecular biological techniques outlined above, however Preferred methods that use the nucleic acids set out above, particularly preferably using a vector shown above and most preferably using one described above Cell.

Your own subject of the invention represent means that are characterized in that they are one above described α-amylase variant contain.

This means every means in which the α-amylase in question in any form for use, i.e. in the desired brought hydrolytic contact to their substrate starch or starch-like polysaccharide or for this is being prepared. The one you want Contact usually takes place in an aqueous environment, which is beneficial is buffered to an appropriate pH and, if necessary more favorable Contains factors. This includes For example, other enzymes that continue the immediate reaction products implement, for example with regard to starch liquefaction for food or Animal feed production or for an ethanol production. These also include low molecular weight Compounds that arise, for example, in oligosaccharides such as including cyclodextrins, or low molecular weight compounds, which further solubilize the cleavage products or favor the overall process Have an effect, such as surfactants in the context of a detergent formulation. It can also be agents in which the desired amylolytic activity only with a big one Time Delay should be induced, such as in temporary adhesive processes, according to which the α-amylase variant added to the adhesive in question early on after a long time by increasing of the water content actually becomes active. This also applies to detergents and cleaning agents, for example to which only after a phase of storage at the moment of dilution in opposite the wash liquor the substrate should become active.

An embodiment of this subject of the invention are such means, which are characterized in that it is detergents or cleaning agents.

The properties according to the invention and important ingredients for such detergents and cleaning agents are described in detail below. At this point, an overview of the most important and therefore particularly preferred embodiments of such washing and detergents.

A washing machine according to the invention is preferably or detergent characterized in that it is 0.000001 weight percent to 5% by weight, and more preferably 0.00001 to 4% by weight, 0.0001 up to 3% by weight, 0.001 to 2% by weight or 0.01 to 1% by weight of the α-amylase variant contains.

A washing machine according to the invention is preferably or cleaning agent, characterized in that it additionally contains other enzymes, in particular hydrolytic enzymes or oxidoreductases, particularly preferably others Amylases, proteases, lipases, cutinases, hemicellulases, cellulases, β-glucanases, Oxidases, peroxidases, laccases.

A washing machine according to the invention is preferably or cleaning agent, characterized in that the α-amylase variant by a the other ingredients of the agent stabilized and / or in your contribution to the washing or cleaning performance of the Is increased by means.

A washing machine according to the invention is preferably or detergent characterized in that it is solid overall, preferably after a compacting step for at least one of the components contained, particularly preferred that it is compacted overall.

A washing machine according to the invention is preferably or cleaning agent characterized in that it is liquid, gel-like or pasty is, preferably encapsulated for at least one of the contained Components, particularly preferably encapsulating at least one of the enzymes contained, very particularly preferably with encapsulation the α-amylase variant.

An important area of application for amylases is that as active components in detergents or cleaning agents for cleaning textiles or solid surfaces, such as for example dishes, floors or tools. In the amylolytic activity serves these applications carbohydrate-containing impurities and especially those starch dissolve hydrolytically or detach from the surface. there can it alone, in suitable media or in detergents or cleaning agents be applied. The conditions to be selected for this, such as Temperature, pH, ionic strength, Redox conditions or mechanical influences should for the respective cleaning problem must be optimized, i.e. with regard to the dirt and on the carrier material. So are usual Temperatures for detergents and cleaning agents in the range of 10 ° C with manual means over 40 ° C and 60 ° C to towards 95 ° at mechanical means or in technical applications. Because with modern Washing machines and dishwashers The temperature is usually continuously adjustable, as are all intermediate levels temperature included. Preferably the ingredients of the funds concerned coordinated. The other conditions can over the rest, outlined below Components of the funds are also very specific to the respective Be designed for cleaning purposes.

Preferred laundry detergents according to the invention and cleaning agents are characterized in that under any of the conditions that can be defined in this way Washing or cleaning performance this agent by adding an α-amylase variant according to the invention to the Recipe is improved without this variant. Synergies are preferred in terms of cleaning performance.

An α-amylase variant according to the invention can both in funds for bulk consumers or technical users as well as in products for private use find, all established and / or appropriate dosage forms in the prior art also embodiments of the present invention. With the washing according to the invention or cleaning agents are all conceivable types of cleaning agents meant both concentrates and agents to be used undiluted; to the Use on a commercial scale, in the washing machine or hand washing, or cleaning. These include detergents, for example for textiles, Carpets, or natural fibers, for according to the present invention, the term detergent is used. This includes for example, dishwashing detergent for dishwashers or manual dishwashing detergent or cleaner for hard surfaces such as metal, glass, porcelain, ceramics, tiles, stone, lacquered Surfaces, Plastics, wood or leather; For according to the present invention, such is the term cleaning agent used. Any type of detergent or cleaning agent represents one embodiment of the present invention, provided that it is an amylase according to the invention is enriched.

Embodiments of the Present Invention encompass all and / or established according to the prior art all appropriate dosage forms of the agents according to the invention. These include for example solid, powdery, liquid, gel or pasty Means, optionally also from several phases, compressed or not compressed; also belong for example: extrudates, granules, tablets or pouches, both in bulk as well as packed in portions.

α-Amylases are combined in agents according to the invention, for example, with one or more of the following ingredients: nonionic, anionic and / or cationic surfactants, bleaching agents, bleach activators, bleaching catalysts, builders and / or cobuilders, solvents, thickeners, sequestering agents, electrolytes, optical brighteners, Graying inhibitors, corrosion inhibitors, in particular silver protection agents, soil release agents, color transfer (or transfer) inhibitors, foam inhibitors, abrasives, dyes, fragrances, antimicrobial agents, UV protection agents, enzymes such as, for example Proteases, (optionally other) amylases, lipases, cellulases, hemicellulases or oxidases, stabilizers, in particular enzyme stabilizers, and other components which are known from the prior art.

The nonionic surfactants used are preferably alkoxylated, advantageously ethoxylated, in particular primary alcohols having preferably 8 to 18 carbon atoms and an average of 1 to 12 moles of ethylene oxide (EO) per mole of alcohol, in which the alcohol residue can be linear or preferably methyl-branched in the 2-position , or can contain linear and methyl-branched radicals in the mixture, as are usually present in oxo alcohol radicals. However, alcohol ethoxylates with linear residues of alcohols of native origin with 12 to 18 carbon atoms, for example from coconut, palm, tallow fat or oleyl alcohol, and an average of 2 to 8 EO per mole of alcohol are particularly preferred. The preferred ethoxylated alcohols include, for example, C _12-14 alcohols with 3 EO or 4 EO, C _9-11 alcohol 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 thereof, 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 averages, which can be an integer or a fraction for a specific product. 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, either as the sole nonionic Surfactant or used in combination with other nonionic surfactants are alkoxylated, preferably ethoxylated or ethoxylated and propoxylated fatty acid alkyl esters, preferably with 1 to 4 carbon atoms in the alkyl chain, in particular Fatty acid methyl ester.

Another class of nonionic surfactants that can advantageously be used are the alkyl polyglycosides (APG). Alkypolyglycosides that can be used satisfy the general formula RO (G) _z , in which R denotes a linear or branched, in particular methyl-branched, saturated or unsaturated, aliphatic radical having 8 to 22, preferably 12 to 18, carbon atoms and G is the Is symbol which stands for a glycose unit with 5 or 6 carbon atoms, preferably for 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. Linear alkyl polyglucosides, ie alkyl polyglycosides, in which the polyglycosyl radical is a glucose radical and the alkyl radical is an n-alkyl radical are preferably used.

Also non-ionic surfactants of the type the amine oxides, for example N-cocoalkyl-N, N-dimethylamine oxide and N-tallow alkyl-N, N-dihydroxyethylamine oxide, and the fatty acid alkanolamides can be suitable. The proportion of these nonionic surfactants is preferably no over that of the ethoxylated fatty alcohols, especially when no longer 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.Other suitable surfactants are polyhydroxy fatty acid amides of the formula (II),

in which RCO stands for an aliphatic acyl radical with 6 to 22 carbon atoms, R ¹ for hydrogen, an alkyl or hydroxyalkyl radical with 1 to 4 carbon atoms and [Z] for a linear or branched polyhydroxyalkyl radical with 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 which R represents a linear or branched alkyl or alkenyl radical having 7 to 12 carbon atoms, R ^{1 represents} a linear, branched or cyclic alkyl radical or an aryl radical having 2 to 8 carbon atoms and R ^{2 represents} a linear, branched or cyclic alkyl radical or an aryl radical or an oxyalkyl radical having 1 to 8 carbon atoms, 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 remainder.

[Z] is preferably obtained by reductive amination of a reducing sugar, for example glucose, fructose, maltose, lactose, galactose, mannose or xylose. The N-alkoxy- or N-aryloxy-substituted compounds can, for example, by reaction with fatty acid methyl esters in counter were an alkoxide as a catalyst in the desired polyhydroxy fatty acid amides.

Anionic surfactants used are, for example, those of the sulfonate and sulfate type. The surfactants of the sulfonate type are preferably C _9-13- alkylbenzenesulfonates, olefin sulfonates, ie mixtures of alkene and hydroxyalkanesulfonates and disulfonates such as are obtained, for example, from C _12-18 monoolefins with a terminal or internal double bond by sulfonation with gaseous sulfur trioxide and subsequent alkaline or acidic hydrolysis of the sulfonation products. Also suitable are alkanesulfonates obtained from C _12-18 alkanes, for example by sulfochlorination or sulfoxidation with subsequent hydrolysis or neutralization. The esters of α-sulfofatty acids (ester sulfonates), for example the α-sulfonated methyl esters of hydrogenated coconut, palm kernel or tallow fatty acids, are also suitable.

Other suitable anionic surfactants are sulfonated fatty acid glycerol esters. Among fatty acid glycerol esters are the mono-, di- and triesters and their mixtures to understand how they in the manufacture by esterification of a monoglycerin with 1 to 3 moles of fatty acid or in the transesterification of triglycerides with 0.3 to 2 moles of glycerin be preserved. Preferred sulfonated fatty acid glycerol esters are included the sulfonation products of saturated fatty acids with 6 to 22 carbon atoms, for example caproic acid, caprylic acid, capric acid, myristic acid, lauric acid, palmitic acid, stearic acid or Behenic acid.

As alk (en) yl sulfates are the alkali and especially the sodium salts of the sulfuric acid half esters of C ₁₂ -C ₁₈ fatty alcohols, for example from 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 this chain length are preferred. Also preferred are alk (en) yl sulfates of the chain length mentioned, which contain a synthetic, petrochemical-based straight-chain alkyl radical which have a degradation behavior similar to that of the adequate compounds based on oleochemical raw materials. The C ₁₂ -C ₁₆ alkyl sulfates and C ₁₂ -C ₁₅ alkyl sulfates as well as C ₁₄ -C ₁₅ alkyl sulfates are preferred from the point of view of washing technology. 2,3-Alkyl sulfates are also suitable anionic surfactants.

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

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

Other anionic surfactants especially soaps. Saturated fatty acid soaps are suitable, like the salts of lauric acid, myristic, palmitic acid, stearic acid, hydrogenated erucic acid and behenic acid and especially from natural ones fatty acids, for example coconut, palm kernel or tallow fatty acids, derived soap mixtures.

The anionic surfactants including the Can soaps in the form of their sodium, potassium or Ammonium salts as well 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 be used in the cleaning agents according to the invention. or detergents in total in an amount of preferably 5% by weight up to 50% by weight, in particular from 8% by weight to 30% by weight on the finished product.

Agents according to the invention can contain bleaching agents. Among the compounds which serve as bleaching agents and supply H ₂ O ₂ in water, sodium percarbonate, sodium perborate tetrahydrate and sodium perborate monohydrate are of particular importance. Further bleaching agents that can be used are, for example, peroxopyrophosphates, citrate perhydrates and H ₂ O ₂ -producing peracidic salts or peracids, such as persulfates or persulfuric acid. The urea peroxohydrate percarbamide can also be used, which can be described by the formula HN ₂ -CO-NH ₂ .H ₂ O ₂ . In particular when using the agents for cleaning hard surfaces, for example in automatic dishwashing, they can, if desired, also contain bleaching agents from the group of organic bleaching agents, although their use is in principle also possible for agents for textile washing. Typical organic bleaching agents are the diacyl peroxides, such as dibenzoyl peroxide. Other typical organic bleaching agents are peroxy acids, examples of which include alkyl peroxy acids and aryl peroxy acids. 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 (phthalimidoxyoxyamide), phthalimidoxybenzoic acid, phthalimidoxybenzoic acid and phthalimidoxybenzoic acid, phthalimidoxybenzoic acid, phthalimidoxybenzoic acid, phthalimidoxybenzoic acid, phthalimidoxybenzoic acid and phthalimidoxybenzoic acid, N-nonenylamidopersuccinates, and aliphatic and araliphatic peroxydicarboxylic acids, such as 1,12-diperoxycarboxylic acid, 1,9-diperoxyazelaic acid, diperoxysebacic acid, diperoxybrassylic acid, the diperoxyphthalic acids, 2-decyldiperoxybutane-1,4-dereo, Noyl aminopercaproic acid) can be used.

The bleach content of the agent can be 1 to 40% by weight and in particular 10 to 20% by weight, where perborate monohydrate or percarbonate is advantageously used becomes. A synergistic use of amylase with percarbonate or of amylase with percarboxylic acid is with the applications WO 99/63036 and WO 99/63037 disclosed.

In order to achieve an improved bleaching effect when washing at temperatures of 60 ° C. and below, and in particular during laundry pretreatment, the agents can also contain bleach activators. Bleach activators which can be used are compounds which, under perhydrolysis conditions, give aliphatic peroxocarboxylic acids having preferably 1 to 10 C atoms, in particular 2 to 4 C atoms, and / or optionally substituted perbenzoic acid. Substances are suitable which carry O- and / or N-acyl groups of the number of carbon atoms mentioned and / or optionally substituted benzoyl groups. Multi-acylated alkylenediamines, in particular tetraacetylethylene diamine (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, are preferred -Tetraacetylglycoluril (TAGU), N-acylimides, especially N-nonanoylsuccinimide (NOSI), acylated phenolsulfonates, especially n-nonanoyl- or isononanoyloxybenzenesulfonate (n- or iso-NOBS), acylated hydroxycarboxylic acids (TOC), such as triethyl-O (triethyl) O, Carboxylic 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, isopropenylacetate, 2,5-diacetoxy-2,5-dihydrofuran and the from DE 196 16 693 and DE 19616 767 known enol esters as well as 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 caprolactam derivatives and / or preferred caprolactam derivatives and / or caprolactam derivatives and / or caprolactam derivatives and / or caprolactam derivatives and / or preferably caprolactam derivatives and / or caprolactam derivatives and / or caprolactam derivatives and / or preferably caprolactam derivatives and / or caprolactam derivatives and / or caprolactam derivatives and / or preferably caprolactam derivatives and / or caprolactam derivatives and / or preferably caprolactam derivatives and actame, for example N-benzoylcaprolactam and N-acetylcaprolactam, 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 hydrophilically substituted acylacetals and those in the German patent application DE 196 16 770 and the international patent application WO 95/14075 described acyllactams are also preferably used. Also those from the German patent application DE 4443 177 known combinations of conventional bleach activators can be used. Nitrile derivatives such as cyanopyridines, nitrile quats, for example N-alkylammonium acetonitrile, and / or cyanamide derivatives can also be used. Preferred bleach activators are sodium 4- (octanoyloxy) -benzenesulfonate, n-nonanoyl- or isononanoyloxybenzenesulfonate (n- or iso-NOBS), undecenoyloxybenzenesulfonate (UDOBS), sodium dodecanoyloxybenzenesulfonate (DOBLOB) -benzene benzo (DOBLOB) -benzenebenzol (DOBSOBO) and decanoic acid (DOBLOB) -benzene benzo OBS 12) and N-methylmorpholinum acetonitrile (MMA). Bleach activators of this type can be used in the customary quantity range from 0.01 to 20% by weight, preferably in amounts from 0.1 to 15% by weight, in particular 1% by weight to 10% by weight, based on the total composition. be included.

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

Agents according to the invention contain in the Usually one or more builders, especially zeolites, silicates, Carbonates, organic cobuilders and - where there are no ecological reasons speak their commitment - too the phosphates. The latter are in particular in cleaning agents for the machine Wash the dishes preferably used builders.

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 is to be mentioned here 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 0164 514 described. preferred Crystalline layered silicates of the formula given are those in which M represents sodium and x the values 2 or 3 accepts. In particular, both β- and δ-sodium disilicate Na ₂ Si ₂ O ₅ .yH ₂ O are preferred. Such compounds are commercially available, for example, under the name ^SKS® (from Clariant). SKS-6 ^{® is} primarily a δ-sodium disilicate with the formula Na ₂ Si ₂ O ₅ .yH ₂ O, while SKS-7 ^{® is} predominantly a β-sodium disilicate. Reaction with acids (for example citric acid or carbonic acid) produces δ-sodium disilicate Kanemit NaHSi ₂ O ₅ .yH ₂ O, commercially available under the names SKS-9 ^® and SKS-10 ^® (from Clariant). It can also be advantageous to use chemical modifications of these layered silicates. For example, the alkalinity of the layered silicates can be suitably influenced. Layered silicates doped with phosphate or carbonate have changed crystal morphologies compared to δ-sodium disilicate, dissolve faster and show increased calcium binding capacity compared to δ-sodium disilicate. Layer silicates are of the general empirical formula x Na ₂ O · y SiO ₂ · z P ₂ O ₅ , in which the ratio x to y is a number from 0.35 to 0.6 and the ratio x to z is a number from 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. Compounds made from crystalline layered silicates with other ingredients can also be used. Compounds with cellulose derivatives, which have advantages in disintegrating action 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, are to be mentioned in particular.

Amorphous sodium silicates with a modulus Na ₂ O: SiO ₂ of 1: 2 to 1: 3.3, preferably 1: 2 to 1: 2.8 and in particular 1: 2 to 1: 2.6, can also be used are delayed in dissolving and have secondary washing properties. The delay in dissolution compared to conventional amorphous sodium silicates can be caused in various ways, for example by surface treatment, compounding, compacting / compression 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 in X-ray diffraction experiments do not provide the sharp X-ray reflections that are typical of crystalline substances, but at most one or more maxima of the scattered X-rays which have a width of several degree units of the diffraction angle. However, it can very well lead to particularly good builder properties if the silicate particles deliver washed-out or even sharp diffraction maxima in electron diffraction experiments. This is to be interpreted as meaning that the products have microcrystalline areas of size 10 to a few hundred nm, values up to max. 50 nm and in particular up to max. 20 nm are preferred. Compacted / compacted amorphous silicates, compounded amorphous silicates and over-dried X-ray amorphous silicates are particularly preferred.

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. However, zeolite X and mixtures of A, X and / or P are also suitable. Commercially available and can preferably be used in the context of the present invention, for example a co-crystallizate of zeolite X and zeolite A (about 80% by weight of zeolite X) ), which is sold by 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; measurement method: Coulter Counter) and preferably contain 18 to 22% by weight, in particular 20 to 22% by weight, of bound water.

Of course, there is also a stake of the well-known phosphates possible as builder substances, provided that such use is not avoided for ecological reasons should be. Among the variety of commercially available The alkali metal phosphates have a particular preference for phosphates Pentasodium respectively Pentapotassium triphosphate (sodium or potassium tripolyphosphate) of greatest importance in the detergent and cleaning agent industry.

Alkali metal phosphates is the general term for the alkali metal (especially sodium and potassium) salts of the various phosphoric acids, in which one can distinguish between metaphosphoric acids (HPO ₃ ) _n and orthophosphoric acid H ₃ PO _{4 in} addition to high molecular weight representatives. The phosphates combine several advantages: They act as alkali carriers, prevent limescale deposits on machine parts or lime incrustations in tissues and also contribute to 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, water-soluble powders that lose water of crystallization when heated and at 200 ° C into the weakly acidic 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). NaH ₂ PO _{4 is} acidic; it arises when phosphoric acid with sodium hydroxide solution on egg a pH of 4.5 is set and the mash is sprayed. Potassium dihydrogen phosphate (primary or monobasic potassium phosphate, potassium biphosphate, KDP), KH ₂ PO ₄ , is a white salt with a density of 2.33 gcm ^-3 , 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 easily water-soluble crystalline salt. It exists anhydrous and with 2 mol. (Density 2.066 gcm ^-3 , water loss at 95 °), 7 mol. (Density 1.68 gcm ^-3 , melting point 48 ° with loss of 5 H ₂ O) and 12 mol. Water ( Density 1.52 gcm ^-3 , melting point 35 ° with loss of 5 H ₂ O), becomes anhydrous at 100 ° and changes to the diphosphate Na ₄ P ₂ O ₇ when heated to a greater extent. Disodium hydrogen phosphate is prepared by neutralizing 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 easily soluble in water.

Trisodium phosphate, tertiary sodium phosphate, Na ₃ PO ₄ , are colorless crystals which, as dodecahydrate, have a density of 1.62 gcm ^-3 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 ), a density of 2.536 gcm ^-3 . Trisodium phosphate is readily soluble in water with an alkaline reaction and is produced by evaporating a solution of exactly 1 mol of disodium phosphate and 1 mol of NaOH. Tripotassium phosphate (tertiary or triphase potassium phosphate), K ₃ PO ₄ , is a white, deliquescent, granular powder with a density of 2.56 gcm ^-3 , has a melting point of 1340 ° and is easily soluble in water with an alkaline reaction. It arises, for example, when Thomas slag is heated with coal and potassium sulfate. Despite the higher price, the more soluble, therefore highly effective, potassium phosphates are often preferred over corresponding sodium compounds in the cleaning agent industry.

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

Condensation of the NaH ₂ PO ₄ or the KH ₂ PO ₄ results in higher molecular weight sodium and potassium phosphates, in which one can differentiate cyclic representatives, the sodium or potassium metaphosphates and chain-like types, the sodium or potassium polyphosphates. A large number of terms are used in particular for the latter: melt or glow phosphates, Graham's salt, Kurrol's and Maddrell's salt. All higher sodium and potassium phosphates are collectively referred to as condensed phosphates.

The technically important pentasodium triphosphate, Na ₅ P ₃ O ₁₀ (sodium tripolyphosphate), is an anhydrous or non-hygroscopic, water-soluble salt of the general formula NaO- [P (O) (ONa) -O] _n that crystallizes with 6 H ₂ O. -Na with n = 3. In 100 g of water about 17 g of the salt of water free of crystal water dissolve at room temperature, about 20 g at 60 ° and about 32 g at 100 °; after heating the solution at 100 ° for two hours, hydrolysis produces about 8% orthophosphate and 15% diphosphate. In the production of pentasodium triphosphate, phosphoric acid is reacted with sodium carbonate solution or sodium hydroxide solution in a stoichiometric ratio and the solution is dewatered by spraying. Similar to Graham's salt and sodium diphosphate, pentasodium triphosphate dissolves many insoluble metal compounds (including lime soaps, etc.). Pentapotassium triphosphate, K ₅ P ₃ O ₁₀ (potassium tripolyphosphate), is commercially available, for example, in the form of a 50% strength by weight solution (> 23% P ₂ O ₅ , 25% K ₂ O). The potassium polyphosphates are widely used in the detergent and cleaning agent industry. There are also sodium potassium tripolyphosphates which can also be used in the context of the present invention. These occur, for example, when hydrolyzing sodium trimetaphosphate with KOH: (NaPO ₃ ) ₃ + 2 KOH → Na ₃ K ₂ P ₃ O ₁₀ + H ₂ O

According to the invention, these are accurate such as sodium tripolyphosphate, potassium tripolyphosphate or mixtures usable 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 are potassium tripolyphosphate and sodium potassium tripolyphosphate usable according to the invention.

Organic cobuilders which can be used in the washing and cleaning agents according to the invention are, in particular, polycarboxylates or polycarboxylic acids, polymeric polycarboxylates, polyaspartic acid, polyacetals, optionally oxidized dextrins, other organic cobuilders (see below) and phosphonates can be used. These classes of substances are described below.

Usable organic builders are, for example, those that can be used in the form of their sodium salts polycarboxylic being under polycarboxylic acids such carboxylic acids can 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), unless such use for ecological reasons 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.

The acids themselves can also be used become. In addition to their builder effect, they typically also have the property of an acidifying component and thus also serve to set a lower and milder one pH of washing or Detergents, unless the mixture of other components Resulting pH desired is. In particular, system-compatible and environmentally compatible 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 be used as pH regulators are used. Such regulators are in the inventive means in amounts of preferably not more than 20% by weight, in particular from 1.2% by weight to 17% by weight.

Polymers are also used as builders Suitable polycarboxylates, these are, for example, the alkali metal salts of polyacrylic acid or polymethacrylic acid, for example those with a molecular weight of 500 to 70,000 g / mol.

In the context of this document, the molecular weights given for polymeric polycarboxylates are weight-average molecular weights 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 to the polymers investigated. This information differs significantly from the molecular weight information for which polystyrene sulfonic acids are used as standard. The molecular weights measured against polystyrene sulfonic acids are generally significantly higher than the molecular weights given in this document.

Suitable polymers are in particular Polyacrylates, which preferably have a molecular weight of 2,000 to 20,000 have g / mol. Because of their superior solubility can from this group, in turn, the short-chain polyacrylates, the molecular weights from 2,000 to 10,000 g / mol, and particularly preferably from 3,000 to 5,000 g / mol, may be preferred.

Copolymers are also suitable Polycarboxylates, especially those of acrylic acid with methacrylic acid and of acrylic acid or methacrylic acid with maleic acid. Copolymers of acrylic acid have been particularly suitable maleic proven to contain 50 to 90 wt .-% acrylic acid and 50 to 10 wt .-% maleic acid. Your relative molecular mass, 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 can be from 0.5 to 20% by weight, in particular 1 to 10% by weight.

To improve water solubility can the polymers also allylsulfonic acids, such as allyloxybenzenesulfonic acid and methallylsulfonic acid, as Contain monomer.

Are also particularly preferred biodegradable polymers from more than two different monomer units, for example those which are salts of acrylic acid and monomers the maleic acid as well as vinyl alcohol or vinyl alcohol derivatives or the salts as monomers of acrylic acid and 2-alkylallylsulfonic acid as well as sugar derivatives.

Other preferred copolymers are those that preferably contain acrolein and acrylic acid / acrylic acid salts as monomers or acrolein and vinyl acetate.

Are also preferred as others Builder substances polymeric aminodicarboxylic acids, their salts or their precursors to call. Polyaspartic acids or are particularly preferred their salts and derivatives.

Other suitable builder substances are polyacetals, which by reacting dialdehydes with polyol carboxylic acids Have 5 to 7 carbon atoms and at least 3 hydroxyl groups can be. Preferred polyacetals are derived from dialdehydes such as glyoxal, glutaraldehyde, Terephthalaldehyde and mixtures thereof and from polyol carboxylic acids such as gluconic and / or glucoheptonic acid receive.

Other suitable organic builder substances are dextrins, for example oligomers or polymers of carbohydrates, which can be obtained by partial hydrolysis of starches. The hydrolysis can be carried out by customary, for example acid or enzyme-catalyzed, processes. They are preferably hydrolysis products with average molecular weights in the range from 400 to 500,000 g / mol. A polysaccharide with a dextrose equivalent (DE) in the range from 0.5 to 40, in particular from 2 to 30, is preferred, DE being a customary measure of the reducing effect of a polysaccharide compared to dextrose, which has a DE of 100 has. Both maltodextrins with a DE between 3 and 20 and dry glucose syrups with a DE between 20 and 37 as well as so-called yellow dextrins and white dextrins with higher molar masses in the range from 2,000 to 30,000 g / mol can be used.

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 97125399, and EP 755 944 ,

Also oxydisuccinates and other derivatives of disuccinates, preferably ethylenediamine disuccinate, are others suitable cobuilders. This is ethylenediamine-N, N'-disuccinate (EDDS) preferably used in the form of its sodium or magnesium salts. Glycerol disuccinates are also preferred in this context and glycerol trisuccinates. Suitable amounts are those containing zeolite and / or formulations containing silicate between 3 and 15 wt .-%.

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

Another class of substances with cobuilder properties represent the phosphonates. These are in particular around hydroxyalkane or aminoalkanephosphonates. Among the Hydroxyalkane phosphonates, 1-hydroxyethane-1,1-diphosphonate (HEDP) is particularly special Meaning as a cobuilder. It is preferably used as the sodium salt the disodium salt being neutral and the tetrasodium salt being alkaline (pH 9) reacts. Preferred aminoalkane phosphonates are ethylenediaminetetramethylenephosphonate (EDTMP), diethylenetriaminepentamethylenephosphonate (DTPMP) and their higher Homologues in question. They are preferably in the form of neutral reactions Sodium salts, for example as the hexasodium salt of EDTMP or used as the hepta and octa sodium salt of DTPMP. As a builder HEDP is preferably used from the class of the phosphonates. The aminoalkanephosphonates also have a pronounced ability to bind heavy metals. Accordingly it may be preferred, especially if the agents also contain bleach be to use aminoalkanephosphonates, in particular DTPMP, or mixtures to use from the phosphonates mentioned.

In addition, all connections that are able to form complexes with alkaline earth ions as cobuilders be used.

Builders can in the agents according to the invention optionally contained in amounts up to 90 wt .-%. they are preferably contained in amounts up to 75 wt .-%. Detergents according to the invention have builder contents of in particular 5% by weight to 50% by weight. In agents according to the invention for the Cleaning hard surfaces, the content is especially for machine cleaning of dishes of builder substances in particular 5% by weight to 88% by weight, where preferably no water-insoluble in such agents Builder materials are used. In a preferred embodiment, agents according to the invention for particularly machine cleaning of dishes, 20% by weight up to 40% by weight more water-soluble organic builder, in particular alkali citrate, 5% by weight to 15 % By weight of alkali carbonate and 20% by weight to 40% by weight of alkali disilicate contain.

Solvent, those in the liquid to gel-shaped Compositions of washing and cleaning agents are used can, come, for example, from the group of mono- or polyhydric alcohols, alkanolamines or glycol ether, provided they are in the specified concentration range are miscible with water. The solvents are preferred 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 ether, 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 and mixtures of these solvents.

solvent can in the liquid up to the invention gel Detergents and cleaning agents in amounts between 0.1 and 20% by weight, but preferably below 15% by weight and in particular below 10 % By weight are used.

Compositions according to the invention can be used to adjust the viscosity one or more thickening or thickening systems added become. These high-molecular substances, which are also swelling agents are usually sucked up and swell the liquids going to eventually in viscous real ones or to go over 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 thickeners come from the groups of natural polymers, the modified natural polymers and the fully synthetic polymers. Such natural polymers are, for example, agar-agar, carrageenan, tragacanth, gum arabic, alginates, pectins, polyoses, guar flour, locust bean gum, starch, dextrins, gelatin and casein. Modified natural substances that are used as thickeners mainly come from the group of modified starches and celluloses. Examples include carboxymethyl cellulose and other cellulose ethers, hydroxyethyl and propyl cellulose and core meal ether. Fully synthetic thickeners are polymers such as polyacrylic and polymethacrylic compounds, vinyl polymers, polycarboxylic acids, polyethers, polyimines, polyamides and polyurethanes.

The thickening can be up to 5% by weight, preferably from 0.05 to 2% by weight, and particularly preferred from 0.1 to 1.5% by weight, based on the finished composition, be included.

The washing or cleaning agent according to the invention can optionally be used as another usual Ingredients sequestrants, electrolytes and other auxiliary substances contain.

Textile detergents according to the invention can be used as optical brightener derivatives of diaminostilbenedisulfonic acid respectively whose alkali metal salts contain. Salts, for example, are suitable 4,4'-bis (2-anilino-4-morpholino-1,3,5-triazinyl-6-amino) stilbene-2,2'-disulfonic acid or similarly structured compounds that replace the morpholino group a diethanolamino group, a methylamino group, an anilino group or carry a 2-methoxyethylamino group. Brighteners can also be used of the type substituted diphenyl styrenes may 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) diphenyl. Mixtures of the aforementioned optical brighteners can also be used become.

Graying inhibitors have that Task, the dirt detached from the textile fiber suspended in the fleet to keep. For this purpose, they are water soluble Suitable colloids mostly of an organic nature, e.g. starch, glue, Gelatin, salts of ether carboxylic acids or ether sulfonic acids of strength or the cellulose or salts of acidic sulfuric acid esters cellulose or starch. Also water soluble, polyamides containing acidic groups are suitable for this purpose. Farther can be used other than the above-mentioned starch derivatives to Example aldehyde starches. Cellulose ethers such as carboxymethyl cellulose (sodium salt) are preferred. Methyl cellulose, hydroxyalkyl cellulose and mixed ethers, such as methyl hydroxyethyl cellulose, Methyl hydroxypropyl cellulose, methyl carboxymethyl cellulose and their Mixtures, for example in amounts of 0.1 to 5 wt .-%, based on the means used.

In order to provide silver corrosion protection, silver corrosion inhibitors can be used in dishwashing detergents according to the invention. Such are known from the prior art, for example benzotriazoles, iron (III) chloride or CoSO ₄ . As for example from the European patent specification EP 0 736084 B1 is known are particularly suitable silver corrosion inhibitors manganese, titanium, zirconium, hafnium, vanadium, cobalt or cerium salts and / or complexes, in which the metals mentioned in one of the oxidation stages II, III , IV, V or VI are present. 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 their mixtures.

Soil release agents or soil repellents are mostly polymers, those when used in a laundry detergent of the laundry fiber dirt-repellent Give properties and / or the dirt-removing ability of the other detergent components support. A comparable effect can also be used when used 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 mixed polymers of polyethylene terephthalate and polyoxyethylene glycol (DT 16 17 141, or DT 22 00 911). The German patent application DT 22 53 063 mentions acidic agents which contain, inter alia, a copolymer of a dibasic carboxylic acid and an alkylene or cycloalkylene polyglycol. Polymers made from ethylene terephthalate and polyethylene oxide terephthalate and their use in detergents are in the German publications DE 28 57 292 and DE 33 24 258 and the European patent specification EP 0 253 567 described. The European patent EP 066 944 relates to agents which contain 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 are known methyl- or ethyl group-end-capped polyesters with ethylene and / or propylene terephthalate and polyethylene oxide terephthalate units and detergents which contain such a soil release polymer. 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 Polyesters are known which, in addition to oxyethylene groups and terephthalic acid units, contain 1,2-propylene, 1,2-butylene and / or 3-methoxy-1,2-propylene groups and glycerol units and are end-capped with C ₁ -C ₄ -alkyl groups are. From the European patent application EP 0 272 033 are known at least partly by C _1-4 alkyl or acyl residues end-capped polyesters with polypropylene terephthalate and polyoxyethylene terephthalate units. The European patent EP 0 274907 describes sulfoethyl end-capped terephthalate-containing soil-release polyesters. According to the European patent application EP 0 357280 Soil-release polyesters with terephthalate, alkylene glycol and poly-C _2-4 glycol units are produced by sulfonation of unsaturated end groups. International patent application WO 95/32232 relates to acidic, aromatic dirt-releasing polyesters. International polymer application WO 97/31085 discloses non-polymeric soil repellent active ingredients for materials made of cotton with several functional units: a first unit, which can be cationic, for example, is capable of adsorption onto the cotton surface by electrostatic interaction, and a second Unit that is made hydrophobic is responsible for the remaining of the active substance at the water / cotton interface.

To those for use in textile detergents according to the invention candidate color transfer inhibitors belong in particular polyvinylpyrrolidones, polyvinylimidazoles, polymers N-oxides such as poly (vinyl pyridine-N-oxide) and copolymers of vinyl pyrrolidone with vinylimidazole.

When used in machine cleaning processes, it may be advantageous to add foam inhibitors to the agents. Suitable 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-like foam inhibitors are, for example, organopolysiloxanes and their mixtures with microfine, optionally silanized silica, and paraffins, waxes, microcrystalline waxes and their mixtures with silanized silica or bistearylethylenediamide. Mixtures of various foam inhibitors are also used with advantages, for example those made of silicone, 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. Mixtures of paraffins and bistearylethylenediamides are particularly preferred.

An inventive cleaning agent for hard surfaces can about it abrasive components, especially from the group including quartz flours, wood flours, plastic flours, chalks and micro glass balls and mixtures thereof. Abrasives are in the cleaning agents according to the invention preferably not over 20 wt .-%, in particular from 5 wt .-% to 15 wt .-%, contain.

Dyes and fragrances are washing and detergents added to the aesthetic impression of the products to improve and the consumer in addition to the washing and cleaning performance a visually and sensory "typical and distinctive "product to disposal to deliver. 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 Examples of the esters are benzyl acetate, phenoxyethyl isobutyrate, p-tert-butylcyclohexyl acetate, linalyl acetate, dimethylbenzylcarbinylacetate, Phenylethyl acetate, linalyl benzoate, benzyl formate, ethyl methylphenyl glycinate, Allyl cyclohexyl propionate, styrallyl propionate and benzyl salicylate. Count among the ethers for example benzyl ethyl ether, for example the aldehydes linear alkanals with 8–18 Carbon atoms, citral, citronellal, citronellyloxyacetaldehyde, cyclamenaldehyde, Hydroxycitronellal, Lilial and Bourgeonal, to the ketones for example the Jonone, α-isomethyl ionone and methyl cedryl ketone, to the alcohols anethole, citronellol, eugenol, Geraniol, linalool, phenylethyl alcohol and terpineol, to the hydrocarbons belong mainly the terpenes like limes and pinene. However, mixtures are preferred different fragrances, which together make an appealing Generate 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. Likewise suitable are muscatel, sage oil, chamomile oil, clove oil, lemon balm oil, mint oil, cinnamon leaf oil, linden blossom oil, juniper berry oil, vetiver oil, olibanum oil, galbanum oil and labdanum oil as well as Orange blossom oil, neroliol, Orange peel oil and sandalwood oil. Usually is the content of detergents and cleaning agents in dyes below 0.01 wt%, while Fragrances can make up up to 2% by weight of the total formulation.

The fragrances can be used directly in the washing and Detergents can be incorporated, but it can also be beneficial be the fragrances on carriers to apply, which increase the adhesion of the perfume to the items to be cleaned and through a slower fragrance release for long-lasting fragrance, in particular of treated textiles. Such carrier materials have, for example Proven cyclodextrins, taking the cyclodextrin perfume complexes additionally can be coated with other auxiliaries. A further preferred carrier for fragrances is the described zeolite X, which instead of or in a mixture can also absorb fragrances with surfactants. Are therefore preferred Detergents and cleaning agents containing the described zeolite X and Fragrances that are preferably at least partially on the zeolite are absorbed.

Preferred dyes, their selection the expert has no difficulty, have a high storage stability and insensitivity to the rest Ingredients of the agents and against light and no pronounced substantivity towards textile fibers, so as not to stain them.

To combat microorganisms, detergents or cleaning agents can contain antimicrobial agents. Depending on the antimicrobial spectrum and mechanism of action, a distinction is made between bacteriostatics and bactericides, fungistatics and fungicides, etc. Important substances from these groups are, for example, benzalkonium chlorides, alkylarylsulfonates, halophenols and phenol mercuric acetate. In the context of the teaching according to the invention, the terms antimicrobial activity and antimicrobial active substance have the customary meaning, as used, for example, by KH Wallhäußer in "Practice of Sterilization, Disinfection-Preservation: Germ Identification-Industrial Hygiene" (5th ed. - Stuttgart; New York Thieme, 1995) Suitable antimicrobial active ingredients are preferably selected from the groups of alcohols, amines, aldehydes, antimicrobial acids or their salts, carboxylic acid esters, acid amides, phenols, phenol derivatives, diphenyls, diphenylalkanes, urea derivatives , Oxygen, nitrogen acetate and formals, benzamidines, isothiazolines, phthalimide derivatives, pyridine derivatives, antimicrobial surface-active compounds, guanidines, antimicrobial amphoteric compounds, quinolines, 1,2-dibromo-2,4-dicyanobutane, iodo-2-propyl-butyl carbamate , Iodine, iodophores, peroxo compounds, halogen compounds and any mixtures of the foregoing.

The antimicrobial active ingredient can be selected from ethanol, n-propanol, i-propanol, 1,3-butanediol, phenoxyethanol, 1,2-propylene glycol, glycerin, undecylenic acid, benzoic acid, salicylic acid, dihydracetic acid, o-phenylphenol, N-methylmorpholinacetonitrile ( MMA), 2-benzyl-4-chlorophenol, 2,2'-methylene-bis- (6-bromo-4-chlorophenol), 4,4'-dichloro-2'-hydroxydiphenyl ether (dichlosan), 2,4,4 '-Trichlor-2'-hydroxydiphenyl ether (trichlosan), chlorhexidine, N- (4-chlorophenyl) -N- (3,4-dichlorophenyl) urea, N, N' - (1,10-decane-diyldi-1- pyridinyl-4-ylidene) bis (1-octanamine) dihydrochloride, N, N'-bis (4-chlorophenyl) -3,12-diimino-2,4,11,13-tetraaza-tetradecanediimidamide, glucoprotamines, antimicrobial surface-active quaternary compounds, guanidines including the bi- and polyguanidines, such as, for example, 1,6-bis (2-ethylhexyl-biguanido-hexane) dihydrochloride, 1,6-di- (N ₁ , N ₁ '-phenyldiguanido- N ₅ , N ₅ ') -hexane-tetrahydochloride, 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 '] hexanes -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-N5, N5') hexane dihydrochloride, omega: omegα-di- (N ₁ , N ₁ '-phenyldiguanido-N ₅ , N ₅ ') -di-n-propylether dihydrochloride, omega: omega '-Di- (N ₁ , N ₁ ' -p-chlorophenyldiguanido-N ₅ , N ₅ ') -di-n-propylether-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-N5, N5 '] hexane dihydrochloride, omega: omega-di- (N ₁ , N ₁ ' -p-chlorophenyl diguanide o-N ₅ , N ₅ ') m-xylene-dihydrochloride, 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 tetrahydrochloride, ethylene bis (1-tolyl biguanide), ethylene bis (p-tolyl biguanide), ethylene bis (3,5-dimethylphenyl biguanide), ethylene bis (p-tert-amylphenyl biguanide) , Ethylene bis (nonylphenyl biguanide), ethylene bis (phenyl biguanide), ethylene bis (N-butylphenyl biguanide), ethylene bis (2,5-diethoxyphenyl biguanide), ethylene bis (2,4-dimethylphenyl biguanide), Ethylene bis (o-diphenyl biguanide), ethylene bis (mixed amyl naphthyl biguanide), N-butyl ethylene bis (phenyl biguanide), trimethylene bis (o-tolyl biguanide), N-butyl trimethyl bis (phenyl biguanide) and the corresponding salts such as acetates, glucon ate, hydrochlorides, hydrobromides, citrates, bisulfates, fluorides, polymaleates, N-Cocosalkylsarcosinate, phosphites, hypophosphites, perfluorooctanoate, silicates, sorbates, salicylates, maleates, tartrates, fumarates, ethylenediamine tetraacetates, iminodiacetates, cinnamates, thiocyanates, arginates, pyromellitates, tetracarboxybutyrates, Benzoates, glutarates, monofluorophosphates, perfluoropropionates and any mixtures thereof. Halogenated xylene and cresol derivatives, such as p-chlorometacresol or p-chloro-meta-xylene, as well as natural antimicrobial active ingredients of vegetable origin (for example from spices or herbs), animal and microbial origin are also suitable. 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 active ingredient 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 arsonium group, peroxo compounds and chlorine compounds are used. Substances of microbial origin, so-called bacteriocins, can also be used.

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

QAV are due to the implementation of tertiary amines with alkylating agents such as methyl chloride, benzyl chloride, Dimethyl sulfate, dodecyl bromide, but also ethylene oxide can be produced. The alkylation of tertiary amines with a long alkyl radical and two methyl groups is particularly successful easy, also the quaternization of tertiary amines with two long ones Residues and a methyl group can be taken with the help of methyl chloride mild conditions become. Amines that over have three long alkyl radicals or hydroxy-substituted alkyl radicals not very reactive and are preferably quaternized with dimethyl sulfate.

Suitable QAC are, for example, benzalkonium chloride (N-alkyl-N, N-dimethyl-benzyl-ammonium chloride, CAS No. 8001-54-5), benzalkon B (m, p-dichlorobenzyl-dimethyl-C12-alkylammonium chloride, CAS No. 58390- 78-6), benzoxonium chloride (benzyl-dodecyl-bis- (2-hydroxyethyl) ammonium chloride), cetrimonium bromide (N-hexadecyl-N, N-trimethyl-ammonium bromide, CAS No. 57-09-0), benzetonium chloride ( N, N-Dimethyl-N- [2- [2- [p- (1,1,3,3-tetramethylbutyl) phenoxy] ethoxy] ethyl] benzylammonium chloride, CAS No. 121-54-0), Dialkyldimethylammonium chlonides such as di-n-decyldimethylammonium chloride (CAS No. 7173-51-5-5), didecyldimethylammonium bromide (CAS No. 2390-68-3), dioctyldimethylammoniumchloric, 1-cetylpyridinium chloride (CAS No . 123-03-5) and thiazoline iodide (CAS No. 15764-48-1) and their mixtures. Particularly preferred QAV are the benzalkonium chlorides with C ₈ -C ₁₈ -alkyl radicals, in particular C ₁₂ -C ₁₄ -alkyl-benzyl-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 Merrell Labs ,

The antimicrobial agents are in amounts of 0.0001% by weight to 1% by weight, preferably 0.001% by weight up to 0.8% by weight, particularly preferably from 0.005% by weight to 0.3% by weight and in particular from 0.01 to 0.2% by weight.

The agents can UV absorbers (UV absorbers) contain that absorb on the treated textiles and the light resistance the fibers and / or the lightfastness of other recipe components improve. Under UV absorbers are organic substances (light protection filters) to understand who are able to absorb ultraviolet rays and the absorbed energy in the form of long-wave radiation, for example Heat again leave.

Compounds which have these desired properties are, for example, the compounds and derivatives of benzophenone which are active by radiationless deactivation and have substituents in the 2- and / or 4-position. Substituted benzotriazoles, 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 urocanoic acid are also suitable. Biphenyl and especially stilbene derivatives such as those in the EP 0728749 A are described and are commercially available as Tinosorb ^® FD or Tinosorb ^® FR ex Ciba. The UV-B absorbers to be mentioned are: 3-benzylidene camphor or 3-benzylidene norcampher and its derivatives, for example 3- (4-methylbenzylidene) camphor, as in the EP 0693471 B1 described; 4-aminobenzoic acid derivatives, preferably 2-ethylhexyl 4- (dimethylamino) benzoate, 2-octyl 4- (dimethylamino) benzoate and amyl 4- (dimethylamino) benzoate; Esters of cinnamic acid, preferably 2-ethylhexyl 4-methoxycinnamate, propyl 4-methoxycinnamate, isoamyl 4-methoxycinnamate, 2-ethylhexyl 2-cyano-3,3-phenylcinnamate (octocrylene); Esters of salicylic acid, preferably salicylic acid 2-ethylhexyl ester, salicylic acid 4-isopropylbenzyl ester, salicylic acid homomethyl ester; 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, for example, 2,4,6-trianilino- (p-carbo-2'-ethyl-1'-hexyloxy) -1,3,5-triazine and octyl triazone, as in US Pat EP 0818450 A1 described or dioctyl butamido triazone (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 in the EP 0694521 B1 described. Also suitable are 2-phenylbenzimidazole-5-sulfonic acid and its alkali, alkaline earth, 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-benzylidene camphor, such as, for example, 4- (2-oxo-3-bornylidene methyl) benzene sulfonic acid and 2-methyl-5- (2-oxo-3-bornylidene) sulfonic acid and their salts.

Derivatives of benzoylmethane, such as 1- (4'-tert-butylphenyl) -3- (4'-methoxyphenyl) propane-1,3-dione, 4-tert-butyl, are particularly suitable as typical UV-A filters -4'-methoxydibenzoylmethane (Parsol 1789), 1-phenyl-3- (4'-isopropylphenyl) propane-1,3-dione and enamine compounds as described in the DE 19712033 A1 (BASF).

The UV-A and UV-B filters can of course also be used in mixtures. In addition to the soluble substances mentioned, insoluble light-protection pigments, namely finely dispersed, preferably nanoized 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. Silicates (talc), barium sulfate or zinc stearate can be used as salts. The oxides and salts are already used in the form of the 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 can have a spherical shape, but it is also possible to use particles which have an ellipsoidal shape or a shape which differs in some other way from the spherical shape. The pigments can also be surface-treated, ie hydrophilized or hydrophobicized. Typical examples are coated titanium dioxides, such as titanium dioxide T 805 (Degussa) or Eusolex ^® T2000 (Merck; silicones and particularly preferably trialkoxyoctylsilanes or simethicones are preferred as hydrophobic coating agents. Micronized zinc oxide is preferably used. Other suitable UV light protection filters are see the overview by P. Finkel in SÖFW-Journal, volume 122 (1996), p. 543.

The UV absorbers are usually in amounts from 0.01% by weight to 5% by weight, preferably from 0.03% by weight up to 1% by weight.

Agents according to the invention can contain further enzymes in addition to the α-amylase variants according to the invention in order to increase the washing or cleaning performance, in principle all enzymes established in the prior art for these purposes can be used. These include in particular proteases, other amylases, lipases, hemicellulases, cellulases or oxidoreductases, and preferably their mixtures. In principle, these enzymes are of natural origin; Based on the natural molecules, improved variants are available for use in detergents and cleaning agents, which are accordingly preferred. Agents ^according to the invention preferably contain enzymes in total amounts of 1 × 10 ^-6 to 5 percent by weight based on active protein. The protein concentration can be determined using known methods, for example the BCA method (bicinchoninic acid; 2,2'-bichinolyl-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 preferred. Examples of this 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 that which can no longer be assigned to the subtilisins in the narrower sense Proteases TW3 and TW7. Subtilisin Carlsberg is available in a further developed form under the trade name Alcalase ^® from Novozymes A / S, Bagsvaerd, Denmark. The subtilisins 147 and 309 are sold under the trade names Esperase ^®, or Savinase ^® from Novozymes. The protease from Bacillus lentus DSM 5483 (WO 91/02792 A1) is derived from the variants listed under the name BLAP ^® , which are described in particular in WO 92/21760 A1, WO 95/23221 A1, WO 02/088340 A2 and PCT / EP02 / 11725 (not yet published). Other usable proteases from various Bacillus sp. and B. gibsonii are based on the as yet 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, which is sold under the trade name Protosol ^® by Advanced Biochemicals Ltd., Thane, India, which is sold under the trade name Wuxi ^® by Wuxi Snyder Bioproducts Ltd., China, and in the trade name 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 amylases which can be used according to the invention are the α-amylases from Bacillus licheniformis, from B. amyloliquefaciens or from B. stearothermophilus and their further developments which are improved for use in detergents and cleaning agents. 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, for this purpose, the α-amylase from Bacillus sp. A 7-7 (DSM 12368) and the cyclodextrin glucanotransferase (CGTase) from B. agaradherens (DSM 9948) described in the application WO 02/44350 A2. It is also possible to use the amylolytic enzymes which belong to the sequence space of α-amylases, which is described in the application WO 03/002711 A2 is defined, and those in the unpublished application DE 10163748 A1 to be discribed. Fusion products of the molecules mentioned can also 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. Another commercial product is the Amylase-LT ^® .

Agents according to the invention can contain lipases or cutinases, in particular because of their triglyceride-cleaving activities, but also in order to generate peracids in situ 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 M-AP ^® and Lipase AML ^® available. For example, the Genencor company can use the lipases or cutinases whose starting enzymes were originally isolated from Pseudomonas mendocina and Fusarium solanii. Other important commercial products, the preparations originally sold by Gist-Brocades 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.

Agents according to the invention can, especially if they for the Treatment of textiles are thought to contain cellulases, each by purpose as pure enzymes, as enzyme preparations or in the form of Mixtures in which the individual components are advantageously complement in terms of their various performance aspects. To count these performance aspects especially contributions for primary washing performance, for secondary washing performance of the agent (anti-redeposition effect or graying inhibition) and finish (tissue effect), up to the exertion of a "stone washed" effect.

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

Agents according to the invention can contain further enzymes, in particular for removing certain problem soils, which are summarized 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 can be found, 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, washing and cleaning agents according to the invention can contain 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 are Denilite ^® 1 and 2 from Novozymes. Advantageously, organic, particularly preferably aromatic, compounds interacting with the enzymes are additionally added in order to increase the activity of the oxidoreductases in question (enhancers) or to ensure the flow of electrons (mediators) in the case of greatly different redox potentials between the oxidizing enzymes and the soiling.

The enzymes used in agents according to the invention either originate originally from microorganisms, for example of the genera Bacillus, Streptomyces, Humicola, or Pseudomonas, and / or are produced by suitable microorganisms according to known biotechnological processes, for example by transgenic expression hosts of the genera Bacillus or filamentous fungi.

Purification of the concerned Enzymes are conveniently made by themselves established processes, for example via precipitation, sedimentation, concentration, Filtration of the liquid Phases, microfiltration, ultrafiltration, exposure to chemicals, Deodorization or suitable combinations of these steps.

Agents according to the invention can Enzymes are added in any form established according to the prior art become. This includes for example by granulation, extrusion or lyophilization obtained solid preparations or, especially with liquid or gel-shaped Means, solutions the enzymes, advantageously if possible concentrated, low in water and / or mixed with stabilizers.

Alternatively, the enzymes can be used for both the solid for as well the liquid Dosage form are 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 as in enclosed in a solidified or in the core-shell type, in which an enzyme-containing core with a water, air and / or Chemical-impermeable Protective layer covered is. Additional active ingredients, for example stabilizers, emulsifiers, pigments, bleaching agents or Dyes are applied. Such capsules are after known methods, for example by shaking or Roll granulation or applied in fluid-bed processes. Are advantageous granules of this type, for example by applying polymeric film formers, low dust and storage stable due to the coating.

It is also possible to have two or assemble several enzymes so that a single Granulate multiple enzyme activities having.

One in an agent according to the invention contained protein and / or enzyme can be particularly during the Storage against damage such as inactivation, denaturation or decay, for example through physical influences, Oxidation or proteolytic cleavage are protected. With microbial Obtaining the proteins and / or enzymes is an inhibition of Proteolysis is particularly preferred, especially if the agents Proteases included. Preferred agents according to the invention contain this Purpose stabilizers.

A group of stabilizers are reversible protease inhibitors. Benzamidine hydrochloride, Borax, boric acids, boronic acids or their salts or esters, including mainly derivatives with aromatic groups, for example ortho-, meta- or para-substituted phenylboronic acids, in particular 4-formylphenyl boronic acid, or the salts or esters of the compounds mentioned. Also peptide aldehydes, that is Oligopeptides with reduced C-terminus, especially those from 2 to 50 monomers are used for this purpose. To the peptidic Reversible protease inhibitors include ovomucoid, among others 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.

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

Lower aliphatic alcohols but above all polyols, such as glycerol, ethylene glycol, Propylene glycol or sorbitol are other frequently used enzyme stabilizers. Di-glycerol phosphate also protects against denaturation by physical influences. 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, among other things, against physical influences or pH fluctuations. Polymers containing polyamine-N-oxide act simultaneously as enzyme stabilizers and as color transfer inhibitors. Other polymeric stabilizers are linear C ₈ -C ₁₈ polyoxyalkylenes. Alkyl polyglycosides can also 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 double function as soil release agents and as enzyme stabilizers. Hydrophobic, nonionic polymer in particular stabilizes any cellulase that may be present.

Reducing agents and antioxidants increase the stability of the enzymes oxidative decay; therefor for example, sulfur-containing reducing agents are common. Other Examples are sodium sulfate and reducing sugars.

Combinations of stabilizers are particularly preferably 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 salts. The action of peptide-aldehyde stabilizers is favorably increased by the combination with boric acid and / or boric acid derivatives and polyols and even more further by the additional action of divalent cations, such as calcium ions.

Agents according to the invention are preferred embodiment characterized in that for example, around the active ingredients contained in time or space release separately, consist of more than one phase. there can be phases in different, but especially phases in the same physical states act.

Agents according to the invention, which are composed of more than one solid component, can be produced in a simple manner by mixing different solid components, in particular powders, granules or extrudates with different ingredients and / or different release behavior, in an overall loose form. Solid agents according to the invention can be prepared from one or more phases in a known manner, for example by spray drying or granulation, the enzymes and any further thermally sensitive ingredients, such as bleaching agents, optionally being added separately later. For the preparation of agents according to the invention with increased bulk density, in particular in the range from 650 g / l to 950 g / l, there is one from the European patent EP 0 486 592 Known method having an extrusion step is preferred. Another preferred production using a granulation process is in the European patent EP 0 642 576 described.

Proteins can be used for solid agents, for example in dried, granulated, encapsulated or encapsulated and additionally dried form can be used. You can use them separately, that is, as your own Phase, or with other ingredients together in the same phase, with or without compacting. Should be microencapsulated Enzymes are processed in solid form, so the water can with processes known from the prior art from the Workup resulting aqueous solutions removed like spray drying, Spin down or by re-solubilization. That way obtained particles usually have a particle size between 50 and 200 µm.

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

At least two solid phases can also be connected to one another. There is thus a possibility of providing a solid agent according to the invention by compressing or compacting it into tablets. Such tablets can be single or multi-phase. This dosage form therefore also offers the possibility of presenting a solid agent according to the invention with two solid phases. For the preparation of agents according to the invention in tablet form, which can be single-phase or multi-phase, single-color or multi-color and / or can consist of several layers, all constituents - if appropriate one layer each - are preferably mixed with one another in a mixer and the mixture by means of conventional tablet presses, for example eccentric presses or rotary presses, with pressing forces in the range from about 50 to 100 kN / cm ² , preferably at 60 to 70 kN / cm ² . In the case of multi-layer tablets in particular, it can be advantageous if at least one layer is pre-compressed. This is preferably carried out at press forces between 5 and 20 kN / cm ² , in particular at 10 to 15 kN / cm ² . A tablet produced in this way preferably has a weight of 10 g to 50 g, in particular 15 g to 40 g. The three-dimensional shape of the tablets is arbitrary and can be round, oval or angular, intermediate forms also being possible.

It is particularly advantageous if in multiphase agents at least one of the phases is an amylase sensitive Material, especially starch contains or is at least partially surrounded or coated by it. In this way, this phase is mechanically stabilized and / or against influences protected from the outside and at the same time over attacked an effective amylase in the wash liquor, so that the release of the Ingredients is facilitated.

Also preferred agents according to the invention are characterized in that they overall liquid, gel or pasty available. The proteins contained, preferably a protein according to the invention, are such means preferably starting from one according to the prior art Technology performed Protein extraction 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 washing or cleaning agents according to the invention in the form of solutions in usual solvents are usually made by simply mixing the ingredients, those in bulk or as a solution can be placed in an automatic mixer.

One embodiment of the present invention is such a liquid, gel-like or pasty Agents to which a protein essential to the invention and / or one of the other proteins and / or one of the other contained ingredients has been encapsulated, preferably in the form of microcapsules. Those with capsules made of amylase-sensitive material are particularly preferred. Such a joint use of amylase-sensitive materials and the amylolytic enzyme essential to the invention in a washing or cleaning agent can show synergy effects, for example in such a way that the starch-splitting enzyme supports the cleavage of the microcapsules and thus controls the release process of the encapsulated ingredients so that their release does not take place during storage and / or not at the beginning of the cleaning process, but only at a certain time. Complex detergent and cleaning agent systems with a wide variety of ingredients and a wide variety of capsule types, which represent particularly preferred embodiments of the present invention, can be based on this mechanism.

A comparable effect is then given when the ingredients of the detergent or cleaning agent spread over at least two different phases, for example two or more solid, interconnected phases of a tablet-like detergent or cleaning agent, or different granules within the same powdery agent. Two or multi-phase cleaners are for use in both machine dishwashers as well as in detergents prior art. The activity of a Amylolytic enzyme in a previously activated phase is a prerequisite for the Activation of a later one Phase, if this is surrounded by an amylase-sensitive envelope or coating or the amylase-sensitive material is an integral part represents the solid phase, in its partial or complete hydrolysis disintegrates the phase in question. The use of what is essential to the invention Enzyme for this is the purpose of a preferred embodiment of the present Invention.

The ingredients of washing and Detergents suitably support each other in their performance. The synergistic use of amylase and color transfer inhibitors for The cleaning performance is increased, for example, with the registration WO 99/63035. It is also known that polymers that coexist can be used as cobuilders, for example alkyl polyglycosides, the activity and stability of the enzymes contained can stabilize and increase, so from application WO 98/45396. It is therefore preferred if one α-Amylase variant according to the invention by one of the rest, listed above Components modified, in particular stabilized and / or in its Contribution to the washing or cleaning performance of the product is increased. Appropriate formulations for agents according to the invention thus represent particularly preferred embodiments of the present Invention.

Within the scope of appropriate means, α-amylase variants according to the invention can serve to activate your own or other phases if they alone or together with at least one other cleaning active or the active ingredient supporting the cleaning effect in one detergents or cleaning agents consisting of more than one phase to be provided. In a corresponding manner, they can also serve Release ingredients from capsules when they or someone else Active ingredient in a detergent or cleaning agent in encapsulated Form is provided.

Another subject of the invention are processes for cleaning textiles or hard surfaces, which are characterized in that in at least one of the process steps an α-amylase variant according to the invention described above becomes active.

Because in this embodiment the invention is realized in that the improved according to the invention enzymatic properties, especially regarding the alkali activity In principle, every cleaning process is used for improvement become. Every cleaning process is enriched by the activity in question, if it is added in at least one process step. such Processes are used, for example, with machines such as common household dishwashers or household washing machines. Preferred procedures are preferred according to the information given above.

Such methods are further preferred, which are characterized in that the α-amylase variant via one described above Means is used.

Any method is particularly preferred, which is characterized in that the α-amylase in the process step concerned used in an amount of 0.01 mg to 400 mg per corresponding process step is, preferably from 0.02 mg to 300 mg, particularly 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 1, particularly preferably 0.005 to 8 mg of the amylolytic protein per 1 wash liquor. The protein concentration can be done using known methods, for example the BCA method (Bicinchonic acid; 2,2'-bichinolyl-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) be determined.

According to the previous versions the present invention is also achieved through the use of α-amylase variants according to the invention realized. Because here too come the favorable properties of the concerned Enzyme to work. These apply in particular to cleaning purposes.

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

It can be the only effective one Components are used. This is preferably done together with at least one other cleaning active or the cleaning effect supportive Active ingredient.

Such is therefore preferred Use, which is characterized in that the α-amylase variant via a agent described above is used.

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

Because this results in the Wash liquor favorably the concentration values given above. This dosage can depending on the cleaning problem from the manufacturer of the product or from the end user be made.

Another embodiment is the use an α-amylase variant according to the invention Treatment of raw materials or intermediate products in textile production , especially for desizing cotton.

Raw materials and intermediates textile production, for example for those based on cotton, are equipped with starch in the course of their manufacture and further processing to enable better processing. This applies to both yarns, intermediate products and textiles the procedures used are called sizing. For removal the simple, that is the starchy protective layer (Desizing, desizing) amylolytic proteins according to the invention are suitable. This is particularly the case when there are alkaline conditions or if other ingredients such as surfactants are present.

Examples

All molecular biological work steps follow standard methods, such as those found in the manual by Fritsch, Sambrook and Maniatis “Molecular cloning: a laboratory manual, "Cold Spring Harbor Laboratory Press, New York, 1989, or equivalent are specified. Enzymes and kits (Kits) were used according to the information of the respective manufacturer.

example 1

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

Starting from a total DNA preparation of B. amyloliquefaciens, as can be obtained under number DSM 7 from the German Collection of Microorganisms and Cell Cultures GmbH, Maschenoder Weg 1b, 38124 Braunschweig (http://www.dsmz.de) , an approximately 1700 bp fragment was obtained via PCR, which contained the 1545 bp gene for the α-amylase. After restriction with BamHI and SpeI, this fragment was ligated into the expression vector pCYTEX P1, which had previously been cut with the same enzymes, whereby the in 3 shown vector pCYTBAA was obtained. An NdeI restriction site was then introduced via the standard site-directed mutagenesis in front 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 shown vector pGBAA-WT was obtained. In principle, any other cloning vector could have been used for the insertion of the α-amylase gene. Finally, sequencing (1.) verified that the orientation of the insert as in 4 shown, ie was correct in relation to the promoter intended for expression, and (2.) the gene still had the completely correct wild-type sequence as described in SEQ ID NO. 3 is shown.

A preparation of this vector pGBAA-WT has now been described in accordance with the description by H. Zhao et al. (1999) "Methods for Optimizing Industrial Enzymes by Directed Evolution" in Industrial Microbiology and Biotechnology; Ed. Demain, AL et al., Washington DC, ASM Press, pages 597-604, is subjected to an error-prone PCR unbalanced nucleotide ratios and the error rate of DNA polymerase increased due to the presence of manganese chloride. The following substances were combined for a 100 μl batch: 10 μl 10 × mutagenesis 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), 50 pmol of each primer (SEQ ID NO. 1 and 2), 5-15 ng of template DNA, a corresponding amount of ddH ₂ O and 3 μl of MnCl ₂ (5 mmol / l). 5 U of commercially available Taq polymerase (Fa QIAGEN, Hilden, Germany) and the approach following the temperature program trained: 60 s denaturation at 95 ° C, 25 cycles with (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.

After the reaction had ended, the PCR product was purified and cloned into suitable cloning and / or expression vectors. In this case, for cleaning the QIAquick ^® PCR Purification Kit (Fa. QIAGEN, Hilden) is selected, and the PCR product was, after restriction with the enzymes NdeI and PstI into the NdeI-PstI restriction site of the vector pGASTON (E. Henke, PhD thesis, Institute for Technical Biochemistry, University of Stuttgart).

To express the BAA variants obtained were initially competent cells from E. coli XL1-blue (available from Stratagene, LaJolla, USA) with the vector pJL3 (MoBi-Tech, Göttingen, Germany) transformed to over the bacteriocin release protein (BRP) is 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 the B. amyloliquefaciens α-amylase mutants after improved alkaline activity of the derived enzyme

Preparation of the enzyme solution

The cells expressing the amylase were disrupted by placing the whole microtiter plate twice in liquid Frozen nitrogen (-196 ° C) and for 45 min at 37 ° C have been thawed. The cell pellet was removed 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.

Conducting the activity test

To carry out a test at pH 7 (standard), 4 Phadebas ^® tablets (cross-linked starch with covalently bound dye) are available in a 50 ml Falcon tube to 30 ml double distilled water (ddH ₂ O); available from Pharmacia & Upjohn GmbH, Diagnostics, Freiburg; Order No. 10-5380-32) and suspended. To carry out at pH 10, 4 Phadebas ^® tablets are suspended as described above, separated from the liquid phase in a glass frit (pore size 1 or 2) and washed twice with 10 ml each of ddH ₂ O. The retentate is resuspended in 30 ml of a 0.1 M glycine / NaON buffer (pH 10) with 0.25 mmol / l CaCl ₂ .

After a good suspension, 150 μl of the suspensions obtained for the respective pH values were pipetted into each well of a MAR5 N50 ^® filter microtiter plate (Millipore, Schwalbach). These were mixed with 50-75 μl 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.) to an underlying second microtiter plate and the absorption of the liquid phases obtained in this way was measured at a wavelength of 620 nm.

In this way, three mutants were created of the α-amylase gene identified: the tribes A42, A29 and B1.

Example 3

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

The identified in this way strains were cultivated according to routine methods with antibiotic selection taken, the pGASTON mutant plasmids in question were isolated and sequenced the deviations from the starting DNA determined.

The resulting DNA and amino acid sequences of the α-amylases BAA A42, BAA A29 and BAA B1 are 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 in the alignment of 2 compared to the wild type sequence.

These variants differ of the wild type of the α-amylase from B. amyloliquefaciens by exchange in individual positions of 13, 32, 194, 197, 203, 230, 297, 356, 406, 414 and 474 in the Counting after SEQ ID NO. 4. Specifically, the following exchanges were determined: L13P, V32A, W194R, S197P, 1203L, A230V, N297D, E356D, K406R, N414S and K474Q.

In detail, BAA A42 has exchanges L13P, W194R, S197P, E356D and N414S, BAA A29 has exchanges L13P, V32A, A230V, N297S, K406R and N414S, and BAA B1 has exchanges L13P, V32A, 1203L, A230V, N297S, K406R, N414S and K474Q. They can therefore also be used instead of as α-amylases BAA A42, A29 and B1
- B. amyloliquefaciens α-amylase L13P / W194R / S197P / E356D / N414S, as
- B. amyloliquefaciens-α-amylase L13P / V32A / A230V / N297S / K406R / N414S, or as
- B. amyloliquefaciens-α-amylase L13P / V32A / 1203L / 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 occurrence. A42 has three additional Exchange in positions 194, 197 and 356 (W194R, S197P and E356D). A29 has four additional ones Exchanges to those in positions 13 and 414, namely in positions 32, 230, 297 and 406 (V32A, A230V, N297S and K406R). Opposite A29 has B1 additionally over two further amino acid substitutions, namely in positions 203 and 474, specifically about the variations I203L and K474Q.

Example 4

Purification and characterization the respective B. amyloliquefaciens amylase variant

Purification of the α-amylase

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

Protein content and activity determination

Accompanying the purification Protein content was determined using the Micro-BCA Protein Assay Kit from Pierce Biotechnology, Rockford, IL, USA (Order No. 23235) and BSA as standard according to the manufacturer. The amylase activities were determined as indicated in Example 2. doing so were average activity yields achieved from 5 to 10%.

Example 5

activity determination

1 unit of amylase activity is defined than the amount of enzyme that produces 1 µmol glucosidic linkages per minute at 37 ° C hydrolyzed.

Phadebas ^® test

A Phadebas ^® tablet is suspended in 10 ml ddH ₂ O. 500 μl aliquots of this are mixed with 50 μl of appropriately diluted sample (activity concentration <1000 U / l) per measurement. After 15 minutes of incubation 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 M NaOH. To determine the blank value, 500 μl of Phadebas suspension are incubated for 15 min at 37 ° C. and, after the immediate addition of 150 μl of 0.5 M NaOH, 50 μl of sample are added. After centrifugation for 2 min at 14,000 rpm (Centrifuge 5417C from Eppendorf, Hamburg), 500 μl of the supernatant are diluted 1: 2 with ddH ₂ O in a cuvette and the absorption at a wavelength of 620 nm is measured against the correspondingly obtained blank value. The activity can be read from a calibration curve previously established with an amylase preparation known activity.

In this way, the specific activities given in Table 1 were determined, which represent a characteristic variable for each enzyme. Table 3: Specific activities of B. amyloliquefaciens amylase variants according to the invention under the conditions of the Phadebas ^® test

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 of the 0.1 M buffer with the appropriate pH are added to 5 ml of the well-suspended substrate suspension. For measurements in the pH range from 4 to 6 acetate buffers, for the range 7 to 8 Tris / HCl buffers, for pH 9 and pH 10 glycine / NaOH buffers and for the measurements at pH 11 carbonate buffers are used. For each pH value, a triple determination is carried out analogously to the protocol for the Phadebas ^® quick test. 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 in comparison to wild-type BAA

This shows that in particular the variant BAA 42 opposite the starting molecule, but also opposite the other variants have a significantly improved alkaline activity. This can only be due to one or both exchanges in the positions 203 and / or 474, specifically 1203L and K474Q.

description of the figures

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

Where:
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

The beginning of mature proteins lies in the count this alignment uniformly at positions 41 and 43.

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

Where:
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 shown in Example 1 the wild type gene of α-amylase from B. amyloliquefaciens (SEQ ID NO. 3) prior to conversion into an expression vector intermediate cloned to an additional Introduce restriction interface.

4 : The expression vector pGBAA-WT

As shown in Example 1 the wild type gene of α-amylase from B. amyloliquefaciens (SEQ ID NO. 3) after the intermediate cloning transferred to this expression vector and this is subjected to an error-prone PCR, as a result of which the BAA variants according to the invention were obtained.

Claims (43)

α-amylase variant with at least two amino acid exchanges with respect to the starting molecule, characterized in that, in position 13, according to the α-amylase count from B. amyloliquefaciens (SEQ ID NO. 4), a proline residue (P) and in position 414 a 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 positions 194, 197 or 356 according to the counting of the α-amylase from B. amyloliquefaciens (SEQ ID NO. 4) is present, preferably in two, particularly preferably in all three of these positions. α-amylase variant according to claim 2, 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. α-amylase variant according to claim 3, characterized in that it is a variant deals with the exchanges 13P / 194R / 197P / 356D / 414S. α-amylase variant according to claim 1, characterized in that in addition at least one amino acid exchange in one of positions 32, 230, 297 or 406 according to the α-amylase count from B. amyloliquefaciens (SEQ ID NO. 4), preferably in two, particularly preferably in three, very particularly preferably in all four of these positions. α-amylase variant according to claim 5, 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 there is an arginine residue in position 406. α-amylase variant according to claim 6, characterized in that it is a variant deals with the exchanges 13P / 32A / 230V / 297D / 406R / 414S. α-amylase variant with at least two amino acid exchanges across from the starting molecule, thereby characterized 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 8, characterized in that in position 203 there is a leucine (L) and / or a position 474 a glutamine (Q) residue. α-amylase variant according to one of the claims 5 to 7, characterized in that in addition at least one amino acid exchange in one of positions 203 or 474 according to the count of the α-amylase from B. amyloliquefaciens (SEQ ID NO. 4) is present, preferably in both positions. α-amylase variant according to claim 10, characterized in that in position 203 there is a leucine (L) and / or a position 474 a glutamine (Q) residue. α-amylase variant according to claim 9 or 11, characterized in that it is a variant with the exchanges 13P / 32A / 203L / 230V / 297D / 406R / 414S / 474Q is. α-amylase variant according to one of claims 1 to 12, characterized in that the starting molecule is derived from an α-amylase which originally comes 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), particularly preferably from B. amyloliquefaciens, B. agaradherens (DSM 9948) or B. sp. A 7-7 (DSM 12368), very particularly from B. amyloliquefaciens. α-amylase variant according to one of the claims 1 to 12, characterized in that the starting molecule is a Hybrid α-amylase acts, 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), particularly 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). α-amylase variant according to claim 14, characterized in that it is in the hybrid α-amylase is one whose partial sequences from the α-amylases from B. amyloliquefaciens and are derived from B. licheniformis. α-amylase variant according to one of the claims 1 to 13, characterized in that it is a variant the α-amylase B. amyloliquefaciens with one of the amino acid exchange combinations L13P / W194R / S197P / E356D / N414S, L13P / V32A / A230V / N297D / K406R / N414S or L13P / V32A / 1203L / A230V / N297D / K406R / N414S / K474Q deals, preferably with the amino acid exchange combination L13P / V1I194R / S197P / E356D / N414S. α-amylase variant according to claim 16, 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). Nucleic acid, the for one of the α-amylase variants according to one of the claims 1 to 17 coded. nucleic acid according to claim 18, characterized in that it is derived from a nucleic acid according to SEQ ID NO. 3 derives. nucleic acid according to claim 19, 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 of these sequences themselves. Vector representing a region with a nucleic acid one of the claims 18 to 20 contains. Vector according to claim 21, characterized in that he is a cloning vector. Vector according to claim 21, characterized in that he is an expression vector. Cell, characterized in that it is a the in the claims 18 to 20 designated nucleic acids contains preferably on a vector according to any one of claims 21 to 23rd Cell according to claim 24, characterized in that she one of the in claims 1 to 17 designated α-amylase variants forms or can be stimulated to form them, preferably using a nucleic acid according to one of the claims 18 to 20, particularly preferably using an expression vector according to claim 23. Cell according to claim 25, characterized in that it is a bacterium, especially one that α-Amylase variant formed secreted into the surrounding medium. Cell according to one of claims 24 to 26, characterized in that that it it is a gram-negative bacterium, preferably of the genera Escherichia coli or Klebsiella, in particular derivatives from E. coli K12, from E. coli B or K. planticola, and very particularly to derivatives of the strains E. coli BL21 (DE3), E. coli RV308, E. coli DH5a, E. coli JM109, E. coli XL-1 or K. planticola (Rf). Cell according to one of claims 24 to 26, characterized in that that it these are gram-positive bacteria, especially of the genera Staphylococcus, Connebacterium or Bacillus, especially of the species Staphylococcus carnosus, Connebacterium glutamicum, Bacillus subtilis, B. alcalophilus, B. licheniformis, B. amyloliquefaciens, B. stearothermophilus, B. agaradherens, B. globigii or B. lentus. A process for producing an α-amylase variant according to any one of claims 1 to 17, preferably using a nucleic acid according to one of claims 18 to 20, particularly preferably using a vector according to one of claims 21 to 23, very particularly preferably using a cell according to one of claims 24 to 28. Agent, characterized in that it is an α-amylase variant according to one of the claims Contains 1 to 17. Agent according to claim 30, characterized in that it a wash or Detergent. Detergent or cleaning agent according to claim 31, characterized characterized it 0.000001% by weight to 5% by weight, in particular 0.00001 to 3% by weight of the α-amylase variant contains. Detergent or cleaning agent according to claim 31 or 32, characterized in that it additional others Contains enzymes especially hydrolytic enzymes or oxidoreductases, especially preferably further amylases, proteases, lipases, cutinases, hemicellulases, Cellulases, β-glucanases, Oxidases, peroxidases, laccases. Detergent or cleaning agent according to one of claims 31 to 33, 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. Detergent or cleaning agent according to one of claims 31 to 34, characterized in that it is total is solid, preferably after a compacting step for at least one of the contained components, particularly preferred that it is total is compacted. Detergent or cleaning agent according to one of claims 31 to 34, characterized in that it is total liquid, gel or pasty is, preferably encapsulated for at least one of the contained Components, particularly preferably encapsulating at least one of the enzymes contained, very particularly preferably with encapsulation the α-amylase variant. Process for cleaning textiles or hard Surfaces, characterized in that in at least one of the process steps an α-amylase variant according to one of claims 1 to 17 becomes active. A method according to claim 37, characterized in that the α-amylase variant via a Agent according to one of the claims 31 to 36 is used. A method according to claim 37 or 38, characterized in that the a-amylase in the relevant process step in an amount of 0.01 mg to 400 mg is used per corresponding process step, preferably from 0.02 mg to 300 mg, particularly preferably from 0.03 mg to 100 mg. Use of an α-amylase variant according to one of the claims 1 to 17 for cleaning textiles or hard surfaces. Use according to claim 40, characterized in that that the α-amylase variant via a Agent according to one of the claims 31 to 36 is used. Use according to claim 40 or 41, characterized in that that per Application, preferably per application in a dishwasher or a washing machine with 0.01 mg to 400 mg of the α-amylase variant are, preferably 0.02 mg to 300 mg, particularly preferably 0.03 mg up to 100 mg. Use of an α-amylase variant according to one of the claims 1 to 17 for the treatment of raw materials or intermediates in textile production, especially for desizing cotton.