WO2020002187A1

WO2020002187A1 - Laccase-containing detergent having improved cleaning power

Info

Publication number: WO2020002187A1
Application number: PCT/EP2019/066589
Authority: WO
Inventors: Nina Mussmann; Susanne Wieland; Inken Prueser; Margret VAN LIER; Christian DEGERING; Ralf G. Berger; Eric ROTTMANN
Original assignee: Henkel Ag & Co. Kgaa
Priority date: 2018-06-28
Filing date: 2019-06-24
Publication date: 2020-01-02
Also published as: DE102018210570A1

Abstract

The invention is in the field of enzyme technology. The invention relates to laccases that are in particular suitable for use in detergents and cleaning agents, all sufficiently similar laccases having a correspondingly similar sequence according to SEQ ID NO:1, and nucleic acids coding for said laccases. The invention further relates to the production of said laccases, to a method for using said laccases, to the use of said laccases as such, and to agents containing said laccases, in particular detergents and cleaning agents.

Description

Laccase detergent with improved cleaning performance

The invention is in the field of enzyme technology. The invention relates to laccases, which can be used in particular with a view to their use in detergents and cleaning agents, all sufficiently similar laccases with a sequence similar to SEQ ID NO: 1 and nucleic acids coding for them. The invention further relates to their production and methods for using these laccases, their use as such and agents containing them, in particular detergents and cleaning agents.

Laccases (EC 1.10.3.2) are copper-containing, "blue" enzymes that are found in many plants, fungi and microorganisms. Laccases are among the oxidoreductases. The catalytically active center contains four copper ions, which can be differentiated according to their spectroscopic properties. The "blue" type 1 copper is involved in the substrate oxidation, one type 2 and two type 3 copper ions form a trinuclear cluster that binds oxygen and reduces it to water. Laccases are also known as p-diphenol oxidases. In addition to diphenols, laccases oxidize many other substrates such as methoxy substituted phenols and diamines. With regard to their substrates, laccases are surprisingly unspecific. Because of their broad substrate specificity and ability to oxidize phenolic compounds, laccases have aroused great interest in industrial applications. Promising areas for the use of laccases include, for example, the delignification and gluing of fiberboard in the wood industry, the dyeing of substances and the detoxification of dyeing waste in the textile industry, and the use in biosensors.

With the help of mediators, i.e. interposed molecules, laccases can also oxidize substrates that they would otherwise not be able to oxidize. The mediators are typically "small molecule compounds" that are oxidized by laccases. The oxidized mediator then in turn oxidizes the actual substrate.

The first laccase was found in the Japanese lacquer tree (Rhus vernicifera) in 1883. Laccases can be found in many plants such as peach, tomato, mango and potato; Laccases are also known from some insects. The most commonly used laccases, however, come from mushrooms, for example from the species Agaricus, Aspergillus, Cerrena, Curvularia, Fusarium, Lentinius, Monocillium, Myceliophtora, Neurospora, Penicillium, Phanerochaete, Phlebia, Pleurotus, Podospora, Schizophyllum, Stagonosporyllum, Sporotosporus. In nature, the function of laccases is, among other things, to participate in the decomposting of lignocellulose, the biosynthesis of cell walls, the browning of fruits and vegetables, and the prevention of microbial attacks on plants. It is often difficult to effectively remove colored stains / stains from soiled laundry or from a soiled object. Strongly colored stains and soiling, ie stains from fruit and / or vegetables, are a particular challenge when removing them. These stains and soiling contain color bodies based on carotenoid compounds, such as a-, b- and g-carotene and lycopene, on porphyrins, such as chlorophyll, and on flavonoid pigments and dye components. This latter group of dye components based on natural flavonoid comprises the strongly colored anthocyanin dyes and pigments based on pelargonidine, cyanidine, delphidine and their methyl esters and the anthoxanthines. These compounds are the origin of most orange, red, purple and blue colors in fruits and are found in all berries, cherries, red and black currants, grapefruits, passion fruit, oranges, lemons, apples, pears, pomegranates, red cabbage, beetroot and also abundant in flowers. Derivatives of cyanidine are present in up to 80% of the pigmented leaves, in up to 70% of the fruits and in up to 50% of the flowers. Specific examples of such soiling include soiling from tea, coffee, red wine, spices such as curry and paprika, orange, tomato, banana, tea, mango, broccoli, carrot, beetroot, spinach and grass. Ballpoint pen ink is also known as very difficult to remove colored stains. These strongly colored flavonoid and carotenoid dyes are often polycyclic and heterocyclic compounds with conjugated double bond systems. This chemical structure is often responsible for the clear color of the compounds.

The difficulties mentioned in the removal of colored stains and soiling are particularly serious when formulating liquid detergents and cleaning agents, since these usually do not contain any bleaching agents. There is therefore a need to further develop liquid detergents and cleaning agents, in particular those without bleach, in such a way that they are improved with regard to the removal of colored stains and soiling, in particular soiling containing anthocyanins.

Surprisingly, it has been found that a hitherto unknown laccase from Marasmiellus palmivorus (Mpa), or sufficiently similar laccases (based on the sequence identity), is particularly suitable for use in liquid detergents or cleaning agents, since it contains a wide range of colored stains and Soiling, in particular anthocyanin-containing soiling, is removed under standard washing conditions.

The invention therefore relates to a laccase comprising an amino acid sequence which has at least 70% sequence identity with the amino acid sequence given in SEQ ID NO: 1 or SEQ ID NO: 2 over its entire length, or variants thereof. The variants according to the invention are characterized in that they consist of a laccase which has an amino acid sequence with at least 70% sequence identity with the amino acid sequence given in SEQ ID NO: 1 or SEQ ID NO: 2 over its entire length, as the starting molecule by one or more conservative ones Amino acid substitution are available. Alternatively or additionally, the variants according to the invention are characterized in that they start from a laccase which has an amino acid sequence with at least 70% sequence identity with the amino acid sequence given in SEQ ID NO: 1 or SEQ ID NO: 2 over its entire length, by fragmentation, Deletion, insertion or substitution mutagenesis are available and comprise an amino acid sequence which has a length of at least 345, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 445, 450, 460, 470, 480, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494 or 495 connected amino acids matches the starting molecule.

Another object of the invention is a method for producing a laccase, comprising providing a starting laccase which has at least 70% sequence identity to the amino acid sequence given in SEQ ID NO: 1 or SEQ ID NO: 2 over its entire length or variants of the starting laccase, wherein the variants are as defined above.

A laccase in the sense of the present patent application therefore includes both the laccase as such and a laccase produced by a method according to the invention. All statements on laccase therefore relate both to laccase as a substance and to the corresponding processes, in particular laccase production processes. A nucleotide sequence corresponding to the amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 2 is given in SEQ ID NO: 3 or SEQ ID NO: 4.

Further objects of the invention are associated with the laccases according to the invention or the production processes for laccases according to the invention for nucleic acids coding for these laccases, non-human host cells according to the invention or nucleic acids containing nucleic acids.

The invention further relates to agents comprising laccases, in particular detergents and cleaning agents, washing and cleaning processes, and uses defined by the laccases described herein, the laccases used here having at least 70% sequence identity with the one in SEQ ID NO: 1 or SEQ ID NO: 2 indicated amino acid sequence over their total length or are variants thereof. The variants used here are characterized in that they consist of a laccase, which has an amino acid sequence with at least 70% sequence identity with the amino acid sequence given in SEQ ID NO: 1 or SEQ ID NO: 2 over its entire length, as a starting molecule by one or more conservative amino acid substitution are available. They are alternative or supplementary Variants used, characterized in that it consists of a laccase which has an amino acid sequence with at least 70% sequence identity with the amino acid sequence given in SEQ ID NO: 1 or SEQ ID NO: 2 over its entire length, as the starting molecule by fragmentation, deletion, insertion or substitution mutagenesis are available and comprise an amino acid sequence that is at least 345, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 485, 486, 487 in length , 488, 489, 490, 491, 492, 493, 494 or 495 connected amino acids matches the starting molecule.

The present invention is based on the surprising finding of the inventors that a basidiomyceta laccase according to the invention, in particular Marasmiellus palmivorus, which comprises an amino acid sequence which is at least 70% identical to the amino acid sequence given in SEQ ID NO: 1 or SEQ ID NO: 2, the removal of colored stains and soiling, in particular soiling containing anthocyanins, under standard washing conditions. This is particularly surprising insofar as the use in detergents or cleaning agents has not been described for any of the basidiomyceta laccases, in particular Marasmiellus palmivorus.

The laccases according to the invention have high stability in detergents or cleaning agents, for example with respect to surfactants and / or bleaching agents and / or with respect to temperature influences and / or with respect to acidic or alkaline conditions and / or with respect to pH changes and / or with respect to denaturing or oxidizing agents Agents and / or against proteolytic degradation and / or against a change in the redox ratios. Consequently, particularly preferred embodiments of the invention provide performance-improved laccase variants. Such advantageous embodiments of laccases according to the invention consequently enable improved washing results on e.g. Soiling containing anthocyanins in a wide temperature range.

A laccase according to the invention has an enzymatic activity, i.e. it is for the oxidation of e.g. capable of phenol, diphenol and other compounds, especially in a detergent. The washing and cleaning agent according to the invention advantageously has an improved cleaning performance, in particular when removing colored stains and soiling. The washing and cleaning agent according to the invention is particularly preferably suitable for removing soiling containing anthocyanins.

Furthermore, a laccase according to the invention is preferably a mature laccase, ie the catalytically active molecule without signal and / or propeptide (s). Unless otherwise stated, the sequences given also relate to mature (processed) enzymes. As used herein, SEQ ID NO: 1 is the amino acid sequence of the mature protein, while SEQ ID NO: 2 indicates the sequence including the signal peptide. "Laccase of the invention" as used herein refers to laccases that are at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81% , 82%, 83%, 84%,

85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91, 5%, 92%, 92.5%, 93%, 93.5%, 94%,

94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 98.6%, 98.7%, 98.8 %, 98.9%,

99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%

Have sequence identity with the amino acid sequence given in SEQ ID NO: 1 or SEQ ID NO: 2 over their entire length or are variants thereof. The variants are characterized in that they can be obtained from a laccase with the specified sequence identity as the starting molecule by one or more conservative amino acid substitution. Alternatively or additionally, the variants used are characterized in that they can be obtained from a laccase with the specified sequence identity as the starting molecule by fragmentation, deletion, insertion or substitution mutagenesis and comprise an amino acid sequence which has a length of at least 345, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494 or 495 related amino acids with the parent molecule matches.

In various embodiments of the invention, the laccase comprises an amino acid sequence which is at least 95%, 95.5%, 96%, 96.5%, 97%, 97.5% of the amino acid sequence given in SEQ ID NO: 1 over its entire length. , 98%, 98.5%, 98.8%, 99.0%, 99.2%, 99.4%, 99.5%, 99.6% or 99.8%.

In other various embodiments of the invention, the laccase is a free enzyme. This means that the laccase can act directly with all components of an agent and, if the agent is a liquid agent, that the laccase is in direct contact with the solvent of the agent according to the invention (e.g. water). In other embodiments, the laccase according to the invention can form an interaction complex with other molecules in an agent or contain an “envelope”. Here, a single or several laccase molecules can be separated from the other constituents of an agent by a structure surrounding them. Such a separating structure can result from, but is not limited to, vesicles, such as a micelle or a liposome. The surrounding structure can also be a virus particle, a bacterial cell or a eukaryotic cell. In various embodiments, the laccase according to the invention can be contained in cells from Basidiomyceta which express this laccase or in cell culture supernatants of such cells.

The identity of nucleic acid or amino acid sequences is determined by a sequence comparison. This sequence comparison is based on the BLAST algorithm established and commonly used in the prior art (cf., for example, Altschul et al. (1990): "Basic local alignment search tool", J. Mol. Biol. 215: 403-410, and Altschul et al. (1997): "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: 3389-3402) and happens principally in that similar sequences of nucleotides or amino acids in the nucleic acid or amino acid sequences are assigned to each other. A tabular assignment of the relevant positions is called alignment. Another algorithm available in the prior art is the FASTA algorithm. Sequence comparisons (alignments), in particular multiple sequence comparisons, are created using computer programs. For example, the Clustal series (cf. e.g. Chenna et al. (2003): "Multiple sequence alignment with the Clustal series of programs", Nucleic Acids Res. 31: 3497-3500), T-Coffee (cf. e.g. Notredame et al. (2000): "T-Coffee: A novel method for multiple sequence alignments", J. Mol. Biol. 302: 205-217) or programs based on these programs or algorithms. Sequence comparisons (alignments) are also possible with the computer program Vector NTI® Suite 10.3 (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California, USA) with the specified standard parameters, whose AlignX module for sequence comparisons is based on ClustalW.

Such a comparison also allows a statement to be made about the similarity of the compared sequences to one another. It is usually expressed in percent identity, i.e. the proportion of identical nucleotides or amino acid residues at the same or in an alignment corresponding positions indicated. The broader concept of homology also includes conserved amino acid exchanges in amino acid sequences, that is, amino acids with similar chemical activity, since these usually have similar chemical activities within the protein. The similarity of the compared sequences can therefore also be given as percent homology or percent similarity. Identity and / or homology information can be made about entire polypeptides or genes or only over individual areas. Homologous or identical regions of different nucleic acid or amino acid sequences are therefore defined by matches in the sequences. Such areas often have identical functions. They can be small and contain only a few nucleotides or amino acids. Such small areas often have essential functions for the overall activity of the protein. It can therefore make sense to relate sequence matches only to individual, possibly small, areas. Unless otherwise stated, identity or homology information in the present application relates to the total length of the nucleic acid or amino acid sequence specified in each case.

In connection with the present invention, the indication that an amino acid position corresponds to a numerically designated position in SEQ ID NO: 1 therefore means that the corresponding position is assigned to the numerically designated position in SEQ ID NO: 1 in an alignment as defined above.

In a further embodiment of the invention, the laccase is characterized in that its cleaning performance is not significantly reduced compared to that of a laccase which comprises an amino acid sequence which corresponds to the amino acid sequences given in SEQ ID NO: 1, ie at least 70%, 75%, 80%, 85%, 90%, 95% of the reference washing performance. The cleaning performance can be determined in a washing system that contains a detergent in a dosage between 4.5 and 7.0 grams per liter of washing liquor as well as the laccase, the laccases to be compared having the same concentration (based on active protein) and the cleaning performance versus Soiling on cotton is determined by measuring the degree of cleaning of the washed textiles. For example, the washing process can take place for 60 minutes at a temperature of 40 ° C. and the water has a water hardness between 5 ° and 25 °, preferably 10 ° and 20 °, more preferably 13 ° and 17 ° and further preferably 15.5 ° and 16. Have 5 ° (German hardness). The concentration of laccase in the detergent intended for this washing system is from 0.001 to 1% by weight, preferably from 0.001 to 0.1% by weight, and more preferably from 0.01 to 0.06% by weight, based on active, purified protein.

A preferred liquid detergent for such a washing system can e.g. be composed as follows (all figures in% by weight): 7% alkylbenzenesulfonic acid, 9% anionic surfactants, 4% Na salts of C12-C18 fatty acids, 7% non-ionic surfactants, 0.7% phosphonates, 3.2 % Citric acid, 3.0% NaOH, 0.04% defoamer, 5.7% 1, 2-propanediol, 0.1% preservatives, 2% ethanol, 0.2% dye transfer inhibitor, remainder demineralized water. The dosage of the liquid detergent is preferably between 4.5 and 6.0 grams per liter of wash liquor, for example 4.7, 4.9 or 5.9 grams per liter of wash liquor. Washing is preferably carried out in a pH range between pH 7.5 and pH 10.5, preferably between pH 7.5 and pH 9.

In the context of the invention, the cleaning performance is determined at 40 ° C. using a liquid detergent as indicated above, the washing process preferably taking place for 60 minutes.

The degree of whiteness, i.e. the lightening of the soiling, as a measure of the cleaning performance, is determined using optical measuring methods, preferably photometrically. A suitable device is, for example, the Minolta CM508d spectrometer. Usually, the devices used for the measurement are calibrated beforehand with a white standard, preferably a supplied white standard.

The use of the respective laccase, which has the same activity, ensures that even if the ratio of active substance to total protein (the values of the specific activity) diverges, the respective enzymatic properties, for example the cleaning performance of certain soiling, are compared. In general, a low specific activity can be compensated for by adding a larger amount of protein. The laccase activity is determined in a customary manner, and preferably by an optical measurement method, preferably a photometric method. The ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) assay is a common assay for determining the activity of laccases, since ABTS is a natural mediator (cf. e.g. Bourbonnais and Paice (1990) FEBS Lett. 267 (1): 99-102, Canas et al. (2010) “Laccases and their natural mediators”, Biotechnol. Advances 28: 694-705, Frasconi et al. (2009) “Kinetic and biochemical properties of high and low redox potential laccases from fungal and plant origin ”Biochemica et Biophysica Acta 1804: 899-908).

The protein concentration can be determined using known methods, e.g. the BCA method (bicinchoninic acid; 2,2'-bichinolyl-4,4'-dicarboxylic acid) or the biuret method (see, for example, Gornall et al. (1948), J. Biol. Chem., 177: 751-766 ) can be determined. In this regard, the determination of the active protein concentration can be carried out by titrating the active centers using a suitable irreversible inhibitor and determining the residual activity (see, for example, Bender et al. (1966), J. Am. Chem. Soc. 88 (24): 5890-5913 ) respectively.

Laccases according to the invention can have further amino acid changes, in particular amino acid substitutions, insertions or deletions, compared to the laccase described in SEQ ID NO: 1 or SEQ ID NO: 2. Such laccases are, for example, by targeted genetic modification, i.e. through mutagenesis processes, further developed and optimized for specific purposes or with regard to special properties (for example with regard to their catalytic activity, stability, etc.). Furthermore, nucleic acids according to the invention can be introduced into recombination approaches and thus used to generate completely new laccases or other polypeptides.

The aim is to introduce targeted mutations such as substitutions, insertions or deletions into the known molecules, for example in order to improve the cleaning performance of enzymes according to the invention. For this purpose, in particular the surface charges and / or the isoelectric point of the molecules and thereby their interactions with the substrate can be changed. For example, the net charge of the enzymes can be changed in order to influence the substrate binding, especially for use in detergents and cleaning agents. Alternatively or additionally, the stability of the enzymes can be increased even further by one or more corresponding mutations and their cleaning performance can thereby be improved. Advantageous properties of individual mutations, e.g. individual substitutions can complement each other. A laccase already optimized with regard to certain properties, e.g. with regard to their activity and / or their tolerance in relation to the substrate spectrum, can therefore be further developed within the scope of the invention.

The following convention is used to describe substitutions that relate to exactly one amino acid position (amino acid exchanges): First, it is natural existing amino acid in the form of the internationally used one-letter code, followed by the associated sequence position and finally the inserted amino acid. Multiple exchanges within the same polypeptide chain are separated from one another by slashes. In the case of insertions, additional amino acids are named after the sequence position. In the case of deletions, the missing amino acid is replaced by a symbol, for example an asterisk or a dash, or a D is given in front of the corresponding position. For example, R45Q describes the substitution of arginine at position 45 by glutamine, N45AQ the insertion of glutamine after the amino acid alanine at position 45 and N45 ^* or DN45 the deletion of asparagine at position 45. This nomenclature is known to the person skilled in the field of enzyme technology.

Another object of the invention is therefore a laccase which is characterized in that it can be obtained from a laccase as described above as the starting molecule by one or more conservative amino acid substitution. The term "conservative amino acid substitution" means the substitution of one amino acid residue for another amino acid residue, this exchange not leading to a change in polarity or charge at the position of the amino acid exchanged, e.g. the exchange of a non-polar amino acid residue for another non-polar amino acid residue. Conservative amino acid substitutions within the scope of the invention include, for example: G = A = S, I = V = L = M, D = E, N = Q, K = R, Y = F, S = T, G = A = I = V = L = M = Y = F = W = P = S = T. The laccase can be placed before and e.g. Even after the conservative amino acid substitution, comprise an amino acid sequence which corresponds to the amino acid sequence given in SEQ ID NO: 1 or SEQ ID NO: 2 over its entire length by at least 70%, 71%, 72%, 73%, 74%, 75%, 76 %, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91 %, 91, 5%, 92%, 92.5%, 93%,

93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 98.6%, 98, 7%,

98.8%, 98.9%, 99.0%, 99, 1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99, 8% or

99.9% is identical.

Alternatively or additionally, the laccase is characterized in that it is obtainable from a laccase as the starting molecule as described above by fragmentation, deletion, insertion or substitution mutagenesis and comprises an amino acid sequence which is at least 345, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494 or 495 related amino acids matches the parent molecule. The laccase can be placed before and e.g. also after the fragmentation, deletion, insertion or substitution mutagenesis comprise an amino acid sequence which is at least 70%, 71%, 72%, 73% of the total length of the amino acid sequence given in SEQ ID NO: 1 or SEQ ID NO: 2, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 90.5%, 91%, 91, 5%, 92%,

92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% . 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% is identical.

For example, it is possible to delete individual amino acids at the termini or in the loops of the enzyme without losing or reducing the catalytic activity. Furthermore, such fragmentation, deletion, insertion or substitution mutagenesis can, for example, also reduce the allergenicity of enzymes and thus overall improve their usability. Advantageously, the enzymes retain their catalytic activity even after mutagenesis, i.e. their catalytic activity corresponds at least to that of the parent enzyme, i.e. in a preferred embodiment, the catalytic activity is at least 80%, preferably at least 90%, of the activity of the starting enzyme. Other substitutions can also have beneficial effects. Both single and multiple contiguous amino acids can be exchanged for other amino acids.

The further amino acid positions are defined here by an alignment of the amino acid sequence of a laccase according to the invention with the amino acid sequence of the basidiomyceta laccase, in particular Marasmiellus palmivorus, as specified in SEQ ID NO: 1 or SEQ ID NO: 2. Furthermore, the assignment of the positions depends on the mature (mature) protein. This assignment is also to be used in particular if the amino acid sequence of a laccase according to the invention comprises a higher number of amino acid residues than the laccase from Basidiomyceta, in particular Marasmiellus palmivorus, according to SEQ ID NO: 1 or SEQ ID NO: 2. Starting from the positions mentioned in the amino acid sequence of the basidiomyceta laccase, in particular Marasmiellus palmivorus, the change positions in a laccase according to the invention are those which are assigned to these positions in an alignment.

Comparative experiments can provide further confirmation of the correct assignment of the amino acids to be changed, that is to say in particular their functional correspondence, according to which the two positions assigned to one another on the basis of an alignment are changed in the same way in both compared laccases and it is observed whether in both the enzymatic activity is changed in the same way. If, for example, an amino acid exchange in a specific position of the basidiomyceta laccase, in particular Marasmiellus palmivorus, according to SEQ ID NO: 1 or SEQ ID NO: 2 is accompanied by a change in an enzymatic parameter, for example by an increase in the « _M value, and becomes one If a corresponding change in the enzymatic parameter, for example also an increase in the _M value, is observed in a laccase variant according to the invention, the amino acid exchange of which has been achieved by the same amino acid introduced, this is to be seen as a confirmation of the correct assignment. In particular, fragments of laccase as defined herein, in particular those according to SEQ ID NO: 1, which are shortened at the N and / or C terminus in such a way that one or more amino acids of laccase, for example 1, 2, 3, 4 , 5, 6, 7, 8, 9 or 10, are no longer included. Of these shortened fragments, variants can also be used in various embodiments of the invention which have the form shortened by (in each case) 1 to 10 N-terminal and / or C-terminal amino acids starting from the amino acid sequence given in SEQ ID NO: 1 the total length at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85 %, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91, 5%, 92%, 92.5%, 93%, 93.5%, 94%,

94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% are identical. For example, according to the invention, laccases are also detected which have an amino acid sequence which, via the laccase, comprises an amino acid sequence which has at least 70%, preferably at least 80%, particularly preferably at least 95% sequence identity with the amino acid sequence given in SEQ ID NO: 1 over its entire length or the variants thereof described here, without the catalytic activity being lost or reduced thereby. Such laccases are preferably those which have N- and / or C-terminal additional amino acids, for example the signal peptide or fragments of the signal peptide, the signal peptide or fragments of the signal peptide being produced in the preparation of the laccase.

According to the invention, laccases are also recorded which have an amino acid sequence which, compared to a laccase comprising an amino acid sequence, has at least 70%, preferably at least 80%, particularly preferably at least 95% sequence identity with the amino acid sequence given in SEQ ID NO: 2 or the one therein described variants thereof are shortened at the N-terminus such that the signal peptide or one or more amino acids of the signal peptide, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, or 22, especially the N-terminal 22 amino acids, are no longer included. Of these laccases with the signal peptide or fragments of the signal peptide, variants can also be used in various embodiments of the invention which have the form shortened by 1 to 22 N-terminal amino acids based on the amino acid sequence given in SEQ ID NO: 2, over the entire length at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91, 5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%,

96.5%, 97%, 97.5%, 98%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99, 1%, 99 , 2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% are identical.

All of the facts mentioned can also be applied to the processes according to the invention for producing a laccase. Accordingly, a method according to the invention comprises a method for producing a laccase, comprising providing a starting laccase which has at least 70%, preferably at least 80%, particularly preferably at least 95% sequence identity to the amino acid sequence given in SEQ ID NO: 1 or SEQ ID NO: 2 over their total length, or variants of the starting laccase, the method according to the invention for producing the variants comprising, for example, one or more of the following process steps: a) introducing one or more conservative amino acid substitution into a starting laccase according to SEQ ID NO: 1 or SED ID NO : 2;

b) modification of the amino acid sequence given in SEQ ID NO: 1 or SED ID NO: 2 by fragmentation, deletion, insertion or substitution mutagenesis such that the laccase comprises an amino acid sequence which is at least 345, 350, 360, 370 in length , 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494 or 495 related amino acids matches the starting molecule ,

All embodiments also apply to the method according to the invention.

In further refinements of the invention, the laccase or the laccase produced by a process according to the invention is still at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91, 5%, 92%, 92 , 5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5 %, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99, 1%, 99.2%, 99.3%, 99.4%, 99.5 %, 99.6%, 99.7%, 99.8% or 99.9% identical to the amino acid sequence given in SEQ ID NO: 1 over its entire length.

Another object of the invention is a previously described laccase which is additionally stabilized, in particular by one or more mutations, for example substitutions, or by coupling to a polymer. An increase in stability during storage and / or during use, for example in the washing process, means that the enzymatic activity lasts longer and thus the cleaning performance is improved. Basically, all stabilization options described and / or useful in the prior art come into consideration. Preference is given to those stabilizations which are achieved via mutations in the enzyme itself, since such stabilizations do not require any further steps following the extraction of the enzyme. Examples of suitable sequence changes are mentioned above. Further suitable sequence changes are known from the prior art.

Stabilization options include:

Protection against the influence of denaturing agents such as surfactants by mutations that change the amino acid sequence on or on the surface of the protein; Exchange of amino acids that are close to the N-terminus for those that presumably come into contact with the rest of the molecule via non-covalent interactions and thus contribute to the maintenance of the globular structure.

Preferred embodiments are those in which the enzyme is stabilized in several ways, since several stabilizing mutations have an additive or synergistic effect.

Another object of the invention is a laccase as described above, which is characterized in that it has at least one chemical modification. A laccase with such a change is called a derivative, i.e. the laccase is derivatized.

In the context of the present application, derivatives are understood to mean those proteins whose pure amino acid chain has been chemically modified. Such derivatizations can be carried out, for example, in vivo by the host cell that expresses the protein. Couplings of low molecular weight compounds such as lipids or oligosaccharides are particularly noteworthy in this regard. However, derivatizations can also be carried out in vitro, for example by chemical conversion of a side chain of an amino acid or by covalent binding of another compound to the protein. For example, the coupling of amines to carboxyl groups of an enzyme to change the isoelectric point is possible. Such a different compound can also be a further protein which is bound to a protein according to the invention, for example via bifunctional chemical compounds. Derivatization is also to be understood to mean covalent binding to a macromolecular carrier or non-covalent inclusion in suitable macromolecular cage structures. For example, derivatizations can influence the substrate specificity or the binding strength to the substrate, or can temporarily block the enzymatic activity if the coupled substance is an inhibitor. This can be useful, for example, for the period of storage. Such modifications can also affect stability or enzymatic activity. They can also serve to reduce the allergenicity and / or immunogenicity of the protein and thus, for example, to increase its skin tolerance. For example, couplings with macromolecular compounds, for example polyethylene glycol, can improve the protein with regard to stability and / or skin tolerance.

Derivatives of a protein according to the invention can also be understood in the broadest sense to mean preparations of these proteins. Depending on the extraction, processing or preparation, a protein can be combined with various other substances, for example from the culture of the producing microorganisms. A protein may also have been specifically mixed with other substances, for example to increase its storage stability. Therefore are according to the invention also all preparations of a protein according to the invention. This is also irrespective of whether it actually exhibits this enzymatic activity in a particular preparation or not. This is because it may be desired that it has little or no activity during storage and that it only develops its enzymatic function at the time of use. This can be controlled, for example, using appropriate accompanying substances. In particular, the joint preparation of laccases with specific inhibitors is possible in this regard.

With regard to all the laccases or laccase variants and / or derivatives described above, those whose catalytic activity and / or their substrate tolerance corresponds to that of the laccase according to SEQ ID NO: 1 are particularly preferred in the context of the present invention, the catalytic activity and the substrate tolerance as determined herein.

The laccases according to the invention and those which can be used in the detergents according to the invention are obtainable from plants, fungi and bacteria. The laccase comprising SEQ ID NO: 1 or SEQ ID NO: 2 can be obtained from Marasmiellus palmivorus.

However, the natural production quantities of laccases are often very low. It may therefore make sense to increase production by expressing laccase genes in foreign production hosts. For this purpose, vectors are generally used which contain a nucleic acid which codes for a laccase according to the invention.

The invention therefore furthermore relates to a nucleic acid which codes for a laccase according to the invention and a vector comprising such a nucleic acid, in particular a cloning vector or an expression vector. In preferred embodiments, the nucleic acid is a nucleic acid according to SEQ ID NO: 3 or SEQ ID NO: 4. Accordingly, a particularly preferred vector according to the invention is a vector which comprises a nucleic acid according to SEQ ID NO: 3 or SEQ ID NO: 4.

These can be DNA or RNA molecules. They can be present as a single strand, as a single strand complementary to this single strand or as a double strand. In the case of DNA molecules in particular, the sequences of both complementary strands must be taken into account in all three possible reading frames. It should also be taken into account that different codons, ie base triplets, can code for the same amino acids, so that a certain amino acid sequence can be encoded by several different nucleic acids. Because of this degeneracy of the genetic code, all nucleic acid sequences which can encode one of the laccases described above are included in this subject matter of the invention. The person skilled in the art is able to determine these nucleic acid sequences without any doubt since, despite the degeneracy of the genetic code, individual codons can be assigned to defined codons. Therefore, the expert can start from a Determine the amino acid sequence for this amino acid sequence coding nucleic acids without problems. Furthermore, one or more codons can be replaced by synonymous codons in the nucleic acids according to the invention. This aspect relates in particular to the heterologous expression of the enzymes according to the invention. Every organism, for example a host cell of a production strain, has a certain codon usage. Codon use means the translation of the genetic code into amino acids by the respective organism. Bottlenecks in protein biosynthesis can occur if the codons lying on the nucleic acid in the organism are opposed to a comparatively small number of loaded tRNA molecules. Although coding for the same amino acid, this means that a codon in the organism is translated less efficiently than a synonymous codon that codes for the same amino acid. Due to the presence of a higher number of tRNA molecules for the synonymous codon, this can be translated more efficiently in the organism. It is possible for a person skilled in the art to use methods which are generally known today, such as chemical synthesis or the polymerase chain reaction (PCR) in conjunction with standard molecular biological and / or protein-chemical methods, to complete the corresponding nucleic acids using known DNA and / or amino acid sequences To produce genes. Such methods are known, for example, from Sambrook, J., Fritsch, EF and Maniatis, T. 2001. Molecular cloning: a laboratory manual, 3rd Edition Cold Spring Laboratory Press.

For the purposes of the present invention, vectors are understood to mean elements consisting of nucleic acids which contain a nucleic acid according to the invention as the characteristic nucleic acid region. They are able to establish this in a species or a cell line as a stable genetic element over several generations or cell divisions. Vectors are special plasmids, in particular circular genetic elements, when used in bacteria. In the context of the present invention, a nucleic acid according to the invention is cloned into a vector. The vectors include, for example, those whose origin is bacterial plasmids, viruses or bacteriophages, or predominantly synthetic vectors or plasmids with elements of very different origins. With the other genetic elements present in each case, vectors can establish themselves as stable units in the host cells concerned over several generations. They can be extrachromosomal as separate units or integrated into a chromosome or chromosomal DNA.

Expression vectors comprise nucleic acid sequences which enable them to replicate in the host cells, preferably microorganisms, particularly preferably bacteria, containing them and to express the nucleic acid contained therein. Expression is influenced in particular by the promoter or promoters which regulate the transcription. In principle, expression can take place through the natural promoter originally located in front of the nucleic acid to be expressed, but also through a promoter of the host cell provided on the expression vector or also through a modified or a completely another promoter of another organism or another host cell. In the present case, at least one promoter is provided for the expression of a nucleic acid according to the invention and used for its expression. Expression vectors can also be regulated, for example by changing the cultivation conditions or when a specific cell density of the host cells containing them has been reached or by adding certain substances, in particular activators of gene expression. An example of such a substance is the galactose derivative isopropyl-β-D-thiogalactopyranoside (IPTG), which is used as an activator of the bacterial lactose operon (lac operon). In contrast to expression vectors, the nucleic acid contained in cloning vectors is not expressed.

The invention furthermore relates to a non-human host cell which contains a nucleic acid or a vector according to the invention or which contains a laccase according to the invention, in particular one which secretes the laccase into the medium surrounding the host cell. A nucleic acid according to the invention or a vector according to the invention is preferably transformed into a microorganism which then represents a host cell according to the invention. Alternatively, individual components, i.e. Nucleic acid parts or fragments of a nucleic acid according to the invention are introduced into a host cell in such a way that the resulting host cell contains a nucleic acid according to the invention or a vector according to the invention. This procedure is particularly suitable when the host cell already contains one or more components of a nucleic acid or a vector according to the invention and the further components are then supplemented accordingly. Methods for transforming cells are established in the prior art and are well known to the person skilled in the art. In principle, all cells are suitable as host cells, i.e. prokaryotic or eukaryotic cells. Preferred are host cells that can be genetically advantageously handled, for example in relation to the transformation with the nucleic acid or the vector and its stable establishment, for example unicellular fungi or bacteria. Preferred host cells are furthermore distinguished by good microbiological and biotechnological manageability. This applies, for example, to easy cultivation, high growth rates, low demands on fermentation media and good production and secretion rates for foreign proteins. Preferred host cells according to the invention secrete the (transgenic) expressed protein into the medium surrounding the host cells. Furthermore, the laccases can be modified by the cells producing them after their production, for example by attaching sugar molecules, formylations, aminations, etc. Such post-translational modifications can functionally influence the laccase.

Further preferred embodiments are those host cells whose activity can be regulated on the basis of genetic regulatory elements which are provided, for example, on the vector, but which may also be present in these cells from the outset. For example, through the controlled addition of chemical compounds that act as activators serve, by changing the cultivation conditions or when reaching a certain cell density, these can be stimulated for expression. This enables economical production of the proteins according to the invention. An example of such a connection is IPTG as described above.

Preferred host cells are prokaryotic or bacterial cells. Bacteria are characterized by short generation times and low demands on the cultivation conditions. As a result, inexpensive cultivation processes or production processes can be established. In addition, the specialist in bacteria in fermentation technology has a wealth of experience. Gram-negative or gram-positive bacteria can be suitable for a special production for various reasons, which can be determined experimentally in individual cases, such as nutrient sources, product formation rate, time requirement etc.

In Gram-negative bacteria such as Escherichia coli, a large number of proteins are secreted into the periplasmic space, i.e. into the compartment between the two membranes that enclose the cells. This can be advantageous for special applications. Furthermore, gram-negative bacteria can also be designed in such a way that they not only express the expressed proteins into the periplasmic space, but also into the medium surrounding the bacteria. Gram-positive bacteria such as Bacilli or Actinomycetes or other representatives of the Actinomycetales, on the other hand, have no outer membrane, so that secreted proteins are immediately released into the medium surrounding the bacteria, usually the nutrient medium, from which the expressed proteins can be purified. They can be isolated directly from the medium or processed further. In addition, gram-positive bacteria are related or identical to most organisms of origin for technically important enzymes and usually form comparable enzymes themselves, so that they have a similar codon use and their protein synthesis apparatus is naturally designed accordingly.

Host cells according to the invention can be changed with regard to their requirements for the culture conditions, have different or additional selection markers or can also express other or additional proteins. In particular, these can also be host cells which transgenically express several proteins or enzymes.

The present invention is in principle applicable to all microorganisms, in particular to all fermentable microorganisms, particularly preferably to those of the genus Bacillus, and leads to the fact that proteins of the invention can be produced by using such microorganisms. Such microorganisms then represent host cells in the sense of the invention.

In a further embodiment of the invention, the host cell is characterized in that it is a bacterium, preferably one which is selected from the group of the genera of Escherichia, Klebsiella, Bacillus, Staphylococcus, Corynebacterium, Arthrobacter, Streptomyces, Stenotrophomonas and Pseudomonas, more preferably one selected from the group of Escherichia coli, Klebsiella planticola, Bacillus licheniformis, Bacillus ientus, Bacillusisusususus, Bacillusisususus, Bacillus amylolillus Bacillus globigii, Bacillus gibsonii, Bacillus clausii, Bacillus halodurans, Bacillus pumilus, Staphylococcus carnosus, Corynebacterium glutamicum, Arthrobacter oxidans, Streptomyces lividans, Streptomyces coelicolor and Stenotrophomonas maltophilia.

The host cell can also be a eukaryotic cell, which is characterized in that it has a cell nucleus. Another object of the invention is therefore a host cell, which is characterized in that it has a cell nucleus. In contrast to prokaryotic cells, eukaryotic cells are able to post-translationally modify the protein formed. Examples include fungi such as Actinomycetes or yeasts such as Saccharomyces or Kluyveromyces. This can be particularly advantageous, for example, if the proteins are to undergo specific modifications in connection with their synthesis which enable such systems. The modifications that eukaryotic systems carry out in particular in connection with protein synthesis include, for example, the binding of low molecular weight compounds such as membrane anchors or oligosaccharides. Such oligosaccharide modifications may be desirable, for example, to reduce the allergenicity of an expressed protein. Co-expression with the enzymes naturally formed by such cells, such as cellulases, can also be advantageous. Furthermore, for example, thermophilic fungal expression systems can be particularly suitable for the expression of temperature-resistant proteins or variants. In preferred embodiments of the invention, the host cell is a basidiomycete cell.

The host cells according to the invention are cultivated and fermented in a conventional manner, for example in discontinuous or continuous systems. In the first case, a suitable nutrient medium is inoculated with the host cells and the product is harvested from the medium after an experimentally determined period. Continuous fermentations are characterized by achieving a steady state in which cells partially die but also regrow over a comparatively long period of time and at the same time the protein formed can be removed from the medium.

Host cells according to the invention are preferably used to produce laccase according to the invention. Another object of the invention is therefore a method for producing a laccase comprising

a) cultivating a host cell according to the invention, and

b) isolating the laccase from the culture medium or from the host cell. This subject of the invention preferably comprises fermentation processes. Fermentation processes are known per se from the prior art and represent the actual large-scale production step, generally followed by a suitable purification method of the product produced, for example the laccase according to the invention. All fermentation processes which are based on a corresponding process for producing a laccase according to the invention represent embodiments of this subject of the invention.

Fermentation processes, which are characterized in that the fermentation is carried out via a feed strategy, are particularly suitable. Here, the media components that are consumed by the ongoing cultivation are fed. As a result, considerable increases can be achieved both in the cell density and in the cell mass or dry mass and / or in particular in the activity of the laccase of interest. Furthermore, the fermentation can also be designed in such a way that undesired metabolic products are filtered out or neutralized by adding buffer or suitable counterions.

The laccase produced can be harvested from the fermentation medium. Such a fermentation process is opposed to isolating the laccase from the host cell, i.e. a product preparation from the cell mass (dry mass) preferred, but requires the provision of suitable host cells or one or more suitable secretion markers or mechanisms and / or transport systems so that the host cells secrete the laccase into the fermentation medium. Alternatively, without secretion, isolation of the laccase from the host cell, i.e. they are purified from the cell mass, for example by precipitation with ammonium sulfate or ethanol, or by chromatographic purification.

All of the facts set out above can be combined to form processes for producing laccases according to the invention.

Surprisingly, it was found that the laccases according to the invention, in particular the laccase comprising SEQ ID NO: 1 or SEQ ID NO: 2, improve the washing performance of liquid detergent formulations in particular and not, as is known from other laccases, the undesired darkening and thus intensification of stains, in particular Stain tea and coffee.

The invention therefore furthermore relates to an agent, in particular a washing and cleaning agent, which contains a laccase as described herein. The laccase has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91, 5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99, 1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity with the amino acid sequence given in SEQ ID NO: 1 or SEQ ID NO: 2 over their entire length or is a variant thereof as described above, which is obtainable as a starting molecule from a laccase described above and before and preferably also after the variation has the specified sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2.

This subject of the invention includes all conceivable types of washing or cleaning agents, both concentrates and agents to be used undiluted, for use on a commercial scale, in the washing machine or for hand washing or cleaning. These include, for example, detergents for textiles, carpets, or natural fibers, for which the term detergent is used. This also includes, for example, dishwashing detergents for dishwashers or manual dishwashing detergents or cleaners for hard surfaces such as metal, glass, porcelain, ceramics, tiles, stone, painted surfaces, plastics, wood or leather, for which the term cleaning agent is used, i.e. in addition to manual and machine Dishwashing detergents, for example, also scouring agents, glass cleaners, toilet scent detergents, etc. The detergents and cleaning agents in the context of the invention also include washing aids which are added to the actual detergent in the case of manual or machine textile washing in order to achieve a further effect. Furthermore, detergents and cleaning agents within the scope of the invention also include textile pre-treatment and post-treatment agents, i.e. agents with which the item of laundry is brought into contact before the actual laundry, for example for dissolving stubborn soiling, and also agents which are contained in one of the actual ones Textile laundry downstream step give the items to be washed further desirable properties such as comfortable grip, freedom from creasing or low static charge. The latter means, among others, the fabric softener counted.

In addition to the laccases mentioned, which are suitable for removing colored stains and soiling, a detergent may contain conventional ingredients which are compatible with this constituent. For example, it can additionally contain one or more color transfer inhibitors, which then preferably contain amounts of 0.1 to 2% by weight, in particular 0.2 to 1% by weight, which in a preferred embodiment are selected from the polymers from vinyl pyrrolidone, vinyl imidazole, vinyl pyridine N-oxide or the copolymers thereof. Both polyvinylpyrrolidones with molecular weights of 15,000 to 50,000 g / mol and polyvinylpyrrolidones with higher molecular weights of, for example, up to over 1,000,000 g / mol, in particular from 1,500,000 to 4,000,000 g / mol, N-vinylimidazole / N- Vinylpyrrolidone copolymers, polyvinyloxazolidones, copolymers based on vinyl monomers and carboxamides, pyrrolidone group-containing polyesters and polyamides, grafted polyamidoamines and Polyethyleneimines, polyamine N-oxide polymers and polyvinyl alcohols. However, enzymatic systems comprising a peroxidase and hydrogen peroxide or a substance which supplies hydrogen peroxide in water can also be used. The addition of a mediator compound for the peroxidase, for example an acetosyringone, a phenol derivative or a phenotiazine or phenoxazine, is preferred in this case, it also being possible to use the above-mentioned polymeric color transfer inhibitor active ingredients. Polyvinylpyrrolidone preferably has an average molar mass in the range from 10,000 to 60,000 g / mol, in particular in the range from 25,000 to 50,000 g / mol. Among the copolymers, those of vinylpyrrolidone and vinylimidazole in a molar ratio of 5: 1 to 1: 1 with an average molar mass in the range from 5000 to 50,000 g / mol, in particular 10,000 to 20,000 g / mol, are preferred.

Detergents and cleaning agents according to the invention, which can be present as powdery solids, in post-compacted particle form, in granular form, as homogeneous solutions or suspensions, can contain, in addition to a laccase according to the invention, all known ingredients customary in such agents, preferably at least one further ingredient in the Means is available. The agents according to the invention can in particular contain surfactants, builders (builders), peroxygen compounds or bleach activators. They can also contain water-miscible organic solvents, further enzymes, sequestering agents, electrolytes, pH regulators and / or further auxiliaries such as optical brighteners, graying inhibitors, foam regulators as well as colorants and fragrances and combinations thereof.

In particular, a combination of a laccase according to the invention with one or more further ingredient (s) of the agent is advantageous, since such agent in preferred embodiments according to the invention has improved cleaning performance due to the resulting synergisms. Such a synergism can be achieved in particular by combining an amylase according to the invention with a surfactant and / or a builder (builder) and / or a peroxygen compound and / or a bleach activator.

Advantageous ingredients of agents according to the invention are disclosed in international patent application WO 2009/121725, beginning there on page 5, penultimate paragraph, and ending on page 13 after the second paragraph. Reference is expressly made to this disclosure and the content of the disclosure therein is included in the present patent application.

The detergent according to the invention can contain additional mediators in order to oxidize the dyes to be removed with greater efficiency. Mediators suitable according to the invention are, for example, Tempo (2,2,6,6-tetramethyl-1-piperidinyloxy), HBT (1-hydroxybenzotriazole), ABTS (2,2'-azinobis-3-ethylbenzothiazole-6-sulphonate), NHA (N -Hydroxy-acetanilide), 2,5-xylidine, ethanol, copper, 4-methylcatechol, N-hydroxyphthalimide, gallic acid, tannic acid, quercetin, syringic acid, Guaiacol, dimethoxybenzyl alcohol, phenol, violuric acid (isonitrosobarbituric acid), phenol red, bromophenol blue, cellulose, p-coumaric acid, rooibos, o-cresol, dichloroindophenol, hydroxybenzotriazole, cycloheximide or vanillin.

In further embodiments of the invention, the agent is characterized in that it

(a) contains 1 to 85% by weight, preferably 5 to 65% by weight, of surfactants; and or

(b) contains 0 to 45% by weight, preferably 0.1 to 15% by weight, of builder (builders); and or

(c) contains 0.0005 to 15% by weight, preferably 0.001 to 5% by weight, of protease; and or

(d) contains 0.0005 to 15% by weight, preferably 0.001 to 5% by weight, lipase; and or

(e) contains 0.00005 to 15% by weight, preferably 0.0001 to 5% by weight, mannanase; and or

(f) contains 0.00005 to 15% by weight, preferably 0.0001 to 5% by weight, cellulase / pectate lyase; and or

(g) 0.00005 to 15 g / washload, preferably 0.0001 to 5 g / washload, contains xanthan lyase; and or

(h) 0.00005 to 15 g / washload, preferably 0.00005 to 15 g / washload, containing endoglucanase capable of digesting xanthan gum.

An agent according to the invention advantageously contains the laccase in an amount from 2 pg to 20 mg, preferably from 5 pg to 17.5 mg, particularly preferably from 20 pg to 15 mg and very particularly preferably from 50 pg to 10 mg per gram of the agent. In addition, the agent according to the invention can advantageously the laccase in an amount of 0.00005 to 15% by weight, based on the active enzyme and the total weight of the agent, preferably from 0.0001 to 5% by weight and particularly preferably from 0.001 to 1 % By weight. The concentration of the laccase in the washing and cleaning agent according to the invention can more preferably be from 0.001 to 0.15% by weight, preferably from 0.005 to 0.06% by weight, based on active protein.

Furthermore, the laccase contained in the agent, and / or further ingredients of the agent, can be coated with a substance that is impermeable to the enzyme at room temperature or in the absence of water, which substance becomes permeable to the enzyme under conditions of use of the agent. Such an embodiment of the invention is thus characterized in that the laccase is coated with a substance impermeable to the laccase at room temperature or in the absence of water. Furthermore, the washing or cleaning agent itself can also be packaged in a container, preferably an air-permeable container, from which it is released shortly before use or during the washing process.

In further embodiments of the invention, the agent is characterized in that it is a) in solid form, in particular as a free-flowing powder with a bulk density of 300 g / l to 1200 g / l, in particular 500 g / l to 900 g / l, or

b) is in pasty or liquid form, and / or c) is in the form of a gel or a sachet (pouch), and / or

d) is in the form of a one-component system, or

e) is divided into several components.

These embodiments of the present invention include all solid, powder, liquid, gel or pasty dosage forms of agents according to the invention, which can optionally also consist of several phases and can be in compressed or uncompressed form. The agent can be in the form of a free-flowing powder, in particular with a bulk density of 300 g / l to 1200 g / l, in particular 500 g / l to 900 g / l or 600 g / l to 850 g / l. The solid dosage forms of the agent also include extrudates, granules, tablets or pouches. Alternatively, the agent can also be liquid, gel-like or pasty, for example in the form of a non-aqueous liquid detergent or a non-aqueous paste or in the form of an aqueous liquid detergent or a water-containing paste. Furthermore, the agent can be in the form of a one-component system. Such funds consist of one phase. Alternatively, an agent can consist of several phases. Such an agent is therefore divided into several components.

Washing or cleaning agents according to the invention can only contain a laccase. Alternatively, they can also contain further hydrolytic enzymes or other enzymes in a concentration appropriate for the effectiveness of the agent. A further embodiment of the invention thus represent agents which further comprise one or more further enzymes. All enzymes which can develop a catalytic activity in the agent according to the invention, in particular a protease, amylase, lipase, cellulase, cutinase, pullulanase, hemicellulase, mannanase, tannase, xylanase, xanthanase, xyloglucanase, β-glucosidase, can preferably be used as further enzymes. Pectinase, carrageenase, perhydrolase, oxidase, oxidoreductase, peroxidase or others. The use of one or more further laccases or multi-copper oxidases in addition to the laccases according to the invention is also possible according to the invention. Further enzymes are advantageously contained in the agent each in an amount of 1 × 10 ⁸ to 5 wt .-% based on active protein. Each additional enzyme is increasingly preferred in an amount of 1 × 10 ⁷ to 3% by weight, from 0.00001 to 1% by weight, from 0.00005 to 0.5% by weight, from 0.0001 to 0.1% by weight and particularly preferably from 0.0001 to 0.05% by weight in agents according to the invention, based on active protein. The enzymes particularly preferably show synergistic cleaning performance against certain soiling or stains, ie the enzymes contained in the composition of the agents mutually support one another in their cleaning performance. Such a synergism is very particularly preferably present between the laccase according to the invention and a further enzyme of an agent according to the invention, including in particular between the laccase mentioned and an amylase and / or a lipase and / or a protease and / or a mannanase and / or a cellulase and / or a pectinase. Synergistic effects can occur not only between different enzymes, but also occur between one or more enzymes and other ingredients of the agent according to the invention.

In the cleaning agents described herein, the enzymes to be used can also be packaged together with accompanying substances, for example from fermentation. In liquid formulations, the enzymes are preferably used as an enzyme liquid formulation (s).

As a rule, the enzymes are not provided in the form of the pure protein, but rather in the form of stabilized, storable and transportable preparations. These prefabricated preparations include, for example, the solid preparations obtained by granulation, extrusion or lyophilization or, particularly in the case of liquid or gel form agents, solutions of the enzymes, advantageously as concentrated as possible, low in water and / or with stabilizers or other auxiliaries.

Alternatively, the enzymes can be encapsulated both for the solid and for the liquid administration form, for example by spray drying or extrusion of the enzyme solution together with a preferably natural polymer or in the form of capsules, for example those in which the enzymes are enclosed as in a solidified gel or in those of the core-shell type in which an enzyme-containing core is coated with a protective layer impermeable to water, air and / or chemicals. Additional active ingredients, for example stabilizers, emulsifiers, pigments, bleaching agents or dyes, can additionally be applied in superimposed layers. Capsules of this type are applied by methods known per se, for example by granular or roll granulation or in fluid-bed processes. Such granules are advantageously low in dust, for example by applying polymeric film formers, and are stable on storage due to the coating.

The enzymes can also be introduced into water-soluble films. Such a film enables the enzymes to be released after contact with water. As used herein, "water soluble" refers to a film structure that is preferably completely water soluble. However, films are also that are substantially water-soluble but have relatively small amounts of a material in the film structure that is not water-soluble; Films with materials that are water-soluble only at relatively high water temperatures or only under restricted pH conditions; and films that include a relatively thin layer of water-insoluble material, all included in the term "water-soluble". Such a film preferably consists of (fully or partially hydrolyzed) polyvinyl alcohol (PVA). However, the film can also contain acid / acrylate copolymers, preferably methacrylic acid / ethyl acrylate copolymer, exclusively or in addition to the PVA, such as that available from Beiland as GBC 2580 and 2600; Styrene-maleic anhydride copolymer (SMA) (available as Scripset (trade name) from Monsanto); Ethylene acrylic acid copolymer (EAA) or metal salt neutralized ethylene Methacrylic acid copolymer (EMAA), known as an ionomer (available from DuPont), in which the acidity of EAA or EMAA is at least about 20 mole percent; Polyether block amide copolymer; Polyhydroxyvaleric acid (available as Biopol (trade name) resins from Imperial Chemical Industries); polyethylene oxide; water soluble polyester or copolyester; Polyethyloxazoline (PEOX 200 from Dow); and water-soluble polyurethane.

Furthermore, it is possible to assemble two or more enzymes together, so that a single granulate has several enzyme activities.

The manufacture of solid detergents and cleaning agents presents no difficulties and can be carried out in a known manner, e.g. by spray drying or granulation, with enzymes and any other thermally sensitive ingredients such as Bleach may be added separately later if necessary. A method having an extrusion step is preferred for producing agents with increased bulk density, in particular in the range from 650 to 950 g / l.

For the preparation of tablets, which can be single-phase or multi-phase, single-color or multi-color and in particular consist of one or more layers, in particular two layers, the procedure is preferably such that all constituents - possibly one layer each - are combined in one Mixer mixed together and the mixture is pressed 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, preferably at 60 to 70 kN. 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, in particular at 10 to 15 kN. In this way, break-resistant tablets, which nevertheless dissolve sufficiently quickly under application conditions, are obtained with breaking and bending strengths of normally 100 to 200 N, but preferably over 150 N. A tablet produced in this way preferably has a weight of 10 to 50 g, in particular 15 to 40 g on. The three-dimensional shape of the tablets is arbitrary and can be round, oval or angular, intermediate forms also being possible. Corners and edges are advantageously rounded. Round tablets preferably have a diameter of 30 to 40 mm. In particular, the size of angular or cuboid tablets, which are mainly introduced via the metering device of the washing machine, depends on the geometry and the volume of this metering device. Exemplary preferred embodiments have a base area of (20 to 30 mm) × (34 to 40 mm), in particular 26 × 36 mm or 24 × 38 mm.

Liquid or pasty agents in the form of solutions containing customary solvents are generally prepared by simply mixing the ingredients, which can be added in bulk or as a solution to an automatic mixer. Solid and / or liquid detergents and cleaning agents according to the invention can, for example, also be packaged in sachets or (preferably self-dissolving) sachets (pouches), in particular also in multi-chamber pouches. For the purposes of the invention, the term liquid also includes any solid state dispersions in liquids. Liquid agents according to the invention can also be multi-phase, the phases can be arranged, for example, vertically, ie one above the other or horizontally, ie next to one another.

Another object of the invention is a method for cleaning textiles or hard surfaces, which is characterized in that an agent according to the invention is used in at least one process step or that a laccase according to the invention becomes catalytically active in at least one process step, in particular in such a way that the laccase in an amount of 40 pg to 4 g, preferably from 50 pg to 3 g, particularly preferably from 100 pg to 2 g and very particularly preferably from 200 pg to 1 g.

In various embodiments, the process described above is characterized in that the laccase is used at a temperature of 0 to 100 ° C., preferably 0 to 60 ° C., more preferably 20 to 45 ° C. and most preferably 40 ° C. becomes.

This includes both manual and machine processes, with machine processes being preferred. Processes for cleaning textiles are generally characterized in that various cleaning-active substances are applied to the items to be cleaned and washed off after the exposure time in several process steps, or in that the items to be cleaned are treated in some other way with a detergent or a solution or dilution of this agent. The simplest form of the process is realized by bringing textiles in need of cleaning into contact with the aqueous liquor, using a conventional washing machine or doing the washing by hand. According to the invention, it is preferred to carry out the process with intensive aeration of the washing liquor, as is the case when using a conventional household machine washing program. The same applies to processes for cleaning all materials other than textiles, especially hard surfaces. All conceivable washing or cleaning processes can be enriched in at least one of the process steps by the use of a washing or cleaning agent according to the invention or a laccase according to the invention and then represent embodiments of the present invention. All the facts, objects and embodiments described for the inventive laccases and agents containing them are also applicable to this subject of the invention. Therefore, reference is expressly made at this point to the disclosure at the appropriate point, with the note that this disclosure also applies to the above method according to the invention. Since laccases according to the invention naturally already have a hydrolytic activity and also develop this in media which otherwise have no cleaning power, such as, for example, in mere buffer, a single and / or the only step of such a method can consist, if desired, of an inventive cleaning component as the only active cleaning component Laccase is brought into contact with the soiling, preferably in a buffer solution or in water. This represents a further embodiment of this subject matter of the invention.

Alternative embodiments of this subject matter of the invention also represent methods for treating textile raw materials or for textile care, in which a laccase according to the invention becomes active in at least one method step. Among these, processes for textile raw materials, fibers or textiles with natural components are preferred, and very particularly for those with wool or silk.

In a further aspect, the present invention relates to the use of a laccase according to the invention or a laccase obtainable by a method according to the invention in a washing or cleaning agent for removing starchy soiling.

Another object of the invention is the use of a laccase according to the invention to improve the washing performance of a washing and cleaning agent, in particular when removing colored stains and soiling, particularly preferably when removing soiling containing anthocyanins.

All facts, objects and embodiments, which are described for laccase according to the invention and agents containing it, are also applicable to the described methods and uses. Therefore, reference is expressly made at this point to the disclosure at the appropriate point with the note that this disclosure also applies to the above-mentioned uses and methods according to the invention.

In a further aspect, the present invention relates to the use of a laccase according to the invention or a laccase obtainable by a method according to the invention or a laccase as used in the agents of the invention described in a washing or cleaning agent for removing anthocyanin-containing soiling. All facts, objects and embodiments that are described for laccase according to the invention and agents containing it can also be applied to this object of the invention. Examples:

The following examples illustrate the invention without, however, restricting it thereto.

Example 1: Identification of the laccase

It was screened in Basidiomycetes for anthocyanin-degrading enzymes. A wild-type enzyme, annotated as laccase, was discovered from Marasmiellus palmivorus (Mpa). This laccase showed a positive wash performance on soils containing anthocyanin without darkening on other soils, which was the case with previously tested enzymes of this class (which is of course undesirable and led to the exclusion of these enzymes from the screening). It opens up many opportunities to increase the genetic and biochemical diversity in the area of enzymes used in detergents.

Example 2: Cultivation

All basidiomycetes were grown in a standard liquid nutrient (SNL) medium. A preculture was first cultivated in SNL medium for one week at 24 ° C and 150 rpm. A standardized inoculum was then transferred to the main culture (SNL). The cultivation was carried out at 24 ° C. and 150 rpm for four days in a reactor system.

The wild-type Mpa laccase was separated by means of AEX centrifuge column chromatography. 19 ml of culture supernatant was added to the Pierce® strand anion exchange spin column (Maxi, equilibrated with 10 ml of 50 mM sodium acetate buffer, pH 5). The columns were then washed twice with 10 ml of 50 mM sodium acetate buffer (pH 5). The laccase was gradually eluted with 10 ml of 2, 4, 6, 8 and 100% 50 mM sodium acetate buffer (pH 5) with 2 M NaCl. The resulting fractions were analyzed for their laccase activity and their bleaching effect on colored textiles.

Example 3: Activity ass

The activity of the laccase was determined using an ABTS assay in the microtiter plate.

The total volume is 300 pl. 245 ml of 50 mM Na acetate buffer (pH 4.5) are mixed with 30 ml of a 5 mM ABTS solution and 10 ml of a 2 mM H ₂ O ₂ solution.

For this, 15 ml of sample are added in a suitable dilution, or 15 ml of 50 mM Na acetate buffer as a blank.

This is followed by a kinetic measurement at 420 nm for 10 min at 30 ° C.

The activity in units is calculated using the following formula:

Test volume = 0.3 ml

Sample volume = 0.015 ml

ABTS = 0.0368 cm ^{2 *} pmo | ^{· 1} , at 405-420 nm The activity in mitioI-released ABTS radicals per minute and ml can then be calculated using the molar extinction coefficient for ABTS:

Activity [pmol * min ¹ * ml ¹ ] = (((DE / minprobe - AE / minBiank) * n * r ² ) / (8ABTs * VT es 0) * (VT est / Vprobe) * Fverdg. Whereby

DE / m inprobe of the absorbance change of the sample per minute,

AE / mineian _k the extinction change of the blank per minute,

R the radius of the MTP well,

e ABTS corresponds to the extinction coefficient of ABTS (0.0368 cm ^{2 *} pmol ¹ , at 405-420 nm),

Viest the test volume,

Vprobe the sample volume and

Fverdg the dilution factor

equivalent.

The activity of the laccase against ABTS is obtained in U / ml. One unit is defined as 1 pmol substrate oxidized per minute under defined conditions.

Example 4: Wash test

A mini-wash test was carried out with the purified wild-type supernatant from Marasmiellus palmivorus, in which the laccase with the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2 is present.

Conditions: 40 ° C, 16 ° dH water, 1 h,

Laccase concentration: 25 mU / ml

stains:

1 . C-S-15 blueberry from CFT

2. C-S-72 Forrest Fruit Ice Cream from CFT

Execution:

• Place punched tissue (diameter = 10 mm) in the microtiter plate, preheat the wash liquor to 40 ° C, final concentration 3, 18 g / l,

• Apply lye and enzyme to the soiling, incubate for 1 h at 40 ° C and 600 rpm,

• then rinse the soiling several times with clear water, let it dry and determine the brightness with a color measuring device. The lighter the fabric, the better the cleaning performance. The L value = brightness is measured here, the higher the brighter.

It is washed with a common liquid detergent without enzymes.

Sample 1: only detergent as a benchmark

Sample 2: Detergent plus laccase from Mpa

So you can clearly see that the laccase performs well on these stains. One speaks of a significant improvement in performance from just 1 unit. As a negative control, the boiled, purified supernatant from Mpa was washed (99 ° C. for 30 min), which shows no washing performance (not shown).

Example 4: Detergent formulation

Claims

claims

1. Laccase comprising an amino acid sequence which has at least 70% sequence identity with the amino acid sequence given in SEQ ID NO: 1 or SEQ ID NO: 2 over its entire length.

2. Laccase or variants thereof, these variants being characterized in that

(a) they are obtainable from a laccase according to claim 1 as the starting molecule by single or multiple conservative amino acid substitution; and or

(b) they are obtainable from a laccase according to claim 1 as a starting molecule by fragmentation, deletion, insertion or substitution mutagenesis and comprise an amino acid sequence which is at least 345, 350, 360, 370, 380, 390, 400, 410 in length , 420, 430, 440, 450, 460, 470, 480, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494 or 495 related amino acids matches the starting molecule.

3. A method for producing a laccase according to claim 1 or 2, comprising providing a starting laccase which has at least 70% sequence identity to the amino acid sequence given in SEQ ID NO: 1 or SEQ ID NO: 2 over its entire length.

4. The method of claim 3, further comprising one or both of the following method steps:

(a) introducing a single or multiple conservative amino acid substitution into the starting laccase;

(b) altering the amino acid sequence of the starting laccase by fragmentation, deletion, insertion or substitution mutagenesis such that the modified laccase comprises an amino acid sequence which is at least 345, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494 or 495 contiguous amino acids corresponds to the starting succase.

5. Nucleic acid coding for a laccase according to claim 1 or 2 or coding for a laccase obtained by a method according to claim 3 or 4.

6. Vector containing a nucleic acid according to claim 5, in particular a cloning vector or an expression vector.

7. A non-human host cell which contains a nucleic acid according to claim 5 or a vector according to claim 6, or which contains a laccase according to claim 1 or 2, or which one laccase obtained by a method according to claim 3 or 4, in particular one which secretes the laccase into the medium surrounding the host cell.

8. A method for producing a laccase comprising

(a) culturing a host cell according to claim 7; and

(b) isolating the laccase from the culture medium or from the host cell.

9. Agent, in particular a washing and cleaning agent, containing at least one laccase according to claim 1 or 2 or a laccase obtained by a method according to claim 3 or 4, the concentration of the at least one laccase being in particular from 0.00005 to 15% by weight. %, preferably from 0.0001 to 5% by weight, more preferably from 0.001 to 1% by weight and particularly preferably from 0.001 to 0.1% by weight, based on active protein.

10. Composition according to claim 9, characterized in that it contains at least one additional mediator consisting of 2,2,6,6-tetramethyl-1-piperidinyloxy, 1-hydroxybenzotriazole, 2,2'-azinobis-3-ethylbenzothiazole -6-sulphonate, N-hydroxyacetanilide, 2,5-xylidine, ethanol, copper, 4-methylcatechol, N-hydroxyphthalimide, gallic acid, tannic acid, quercetin, syringic acid, guaiacol, dimethoxybenzyl alcohol, phenol, violuric acid, phenol red, bromophenol blue, Cellulose, p-coumaric acid, rooibos, o-cresol, dichloroindophenol, hydroxybenzotriazole, cycloheximide or vanillin group is selected.

1 1. A method for cleaning textiles or hard surfaces, characterized in that in at least one process step, an agent according to claim 9 or 10 is used, or that in at least one process step a laccase according to claim 1 or 2 or one according to a method according to claim 3 or 4 obtained laccase becomes catalytically active.

12. Use of a laccase according to claim 1 or 2 or a laccase obtained by a process according to claim 3 or 4 in a washing or cleaning agent for removing moving stains and soiling, in particular anthocyanin-containing soiling.