NO176404B - Detergent additive comprising a peroxidase and hydrogen peroxide, a precursor for hydrogen peroxide or an enzymatic system capable of developing hydropower peroxide, for bleaching textile, detergent composition - Google Patents

Description

1

The present invention relates to a detergent additive

which comprises an enzyme system with the ability to exert a bleaching effect on textiles during washing processes, a detergent mixture containing the detergent additive as well as the use of the detergent additive or the detergent mixture.

The use of bleaching agents in washing processes and as components in detergent mixtures is well known in the field. Thus, bleaching agents are incorporated into or sold as components in a large part of the commercially available detergent mixtures. Traditionally, important bleaching agents that are incorporated into detergent mixtures are compounds that act as precursors for hydrogen peroxide that is formed during the washing process. Perborates and per-carbonates are the most important examples of compounds which are used as detergent additives and which exert a bleaching effect in this way. The detailed mechanism of perborate and percarbonate bleaching is currently not known, but it is generally assumed that hydrogen peroxide converts colored substances that are responsible for the coloration of textiles into uncolored materials by oxidation and that some oxidation of the colored substances can also take place due to their direct interaction with perborate or percarbonate.

Although these traditional bleaching systems seem very satisfactory in many cases and have been widely accepted, many attempts have been made to improve their performance over the years. One significant goal has been to provide bleaching systems that work at low temperatures and are generally more efficient than the traditional systems. It has also been an important goal to develop systems that lead to a better utilization of a bleaching agent incorporated in detergent mixtures or otherwise used for bleaching textiles. Among other things, these developments have been accelerated by the desire of the consumer to carry out washing at low temperatures that are not optimal for the functioning of the traditional bleaching systems, where the optimal operating temperature is at least 60°C, and by the environmental aspects with regard to to release, to the environment, large quantities of bleaching agents added in excess amounts to detergent mixtures to achieve adequate bleaching, but which are not fully utilized during the washing processes. In connection with these attempts to develop improved bleaching systems, considerable attention has been directed to the possibility of developing powerful and effective bleaching substances during washing using hydrogen peroxide from its traditional precursors, e.g. perborate and percarbonate. An example of successful development along these lines is the TAED (tetra-acetyl-ethylenediamine)-based system now in widespread use in detergent formulations (see, e.g., Second World Conference of Detergents, A.R. Baldwin (publisher), American Oil Chemists<1> Society, 1987,

pp. 177-180.

It has surprisingly been found possible to enhance the bleaching effect of hydrogen peroxide during washing processes by using a group of enzymes where hydrogen peroxide is used as a substrate for the oxidation of organic or inorganic substances (such enzymes are usually called peroxidases).

Consequently, the present invention relates to a detergent additive with the ability to exert a bleaching effect on textiles and which comprises an enzyme which exhibits a peroxidase effect. The detergent additive according to the invention will also comprise hydrogen peroxide or a precursor for hydrogen peroxide, preferably perborate or percarbonate. In this context, the expression "enzyme exhibiting peroxidase effect" is understood to indicate an enzyme with a mode of action similar to that of peroxidase, and will be used synonymously with it. Incidentally, reference is made to requirement 1.

It is well known in the art (see, eg, B.C. Saunders et al., Peroxidase, London, 1964, p. 10 et seq.) that peroxidases act on various amino and phenolic compounds resulting in the production of a color. Against this background, it must be considered surprising that peroxidases can also exert a bleaching effect on colored substances. While the mechanism governing the ability of peroxidases to bleach stains on textiles caused by such colored substances has not yet been elucidated, it is currently believed that these systems work by reducing hydrogen peroxide (substrate 1) and oxidizing the colored substance in the stain (substrate 2), whereby a colorless substance is developed, and as a result the stain is removed from the textile. This reaction is shown in reaction scheme 1 below.

Reaction scheme 1;

Donor (substrate 2) + H202 -+ oxidized donor + 2 H20.

As the peroxidase will generally show affinity for colored substances present in the stain which act as the substrate for the enzyme (this will typically be natural dyes, e.g. various polyphenols), the bleaching will be targeted towards the stains, for which reason a more efficient utilization of hydrogen peroxide by using peroxidase for the bleaching with the conventional bleaching methods where an excess of hydrogen peroxide must be present to achieve an adequate bleaching effect.

This means that it may be possible, by using the detergent additives according to the invention, to bleach stains on textiles, and reduce the amount of hydrogen peroxide or precursors thereof in the additive or in the detergent mixtures containing the additive, and still provide a satisfactory bleaching effect. Thus, the environmental burden, that is to say the amount of unused bleach released into the environment, can be reduced when a detergent additive according to the invention is used for bleaching. Furthermore, as a smaller amount of hydrogen peroxide needs to be added to achieve effective bleaching, the overall performance of detergent compositions in which such bleaching agents are included (the performance of e.g. detergent enzymes tends to deteriorate when large amounts bleach is used) be improved.

It is further included here that peroxidases with specificity for a particular class of colorant (e.g. betalains, carotenoids, flavonoids) can be used for special purposes, while a peroxidase with low specificity (that is, acting on several natural colorants) can is used for a more non-specific enzymatic bleaching.

It has previously been reported that peroxidases can decolorize certain pigments (see, for example, W. Schreiber, Biochem. Biophys. Res. Commun. 63 (2), 1975, pp. 509-514, where the breakdown of 3-hydroxyflavone is described by horseradish peroxidase, A. Ben Aziz, Phytochemistry 10, 1971, pp. 1445-1452, which describes the bleaching of carotene by peroxidase, and B. P. Wasserman, J. Food Sci.

49, 1984, pp. 536-538, which describes the decolorization of betalain by horseradish peroxidase). All these publications describe test methods whereby the pigment in question is incubated together with the enzyme in solution. Therefore, they do not in any way indicate that peroxidases can be effective for bleaching normally dry stains on textiles in which the colored substance(s) may have been absorbed or partially absorbed into the fibers of the textile so that they become less accessible to enzymatic action and/ or in which the colored substances may have been oxidized by atmospheric oxygen so that actually a different kind of colored substance than that present in a solution of the dye may have been produced.

Furthermore, Ben Aziz et al. and Wasserman et al. the bleaching effect of peroxidase on carotene and betalain respectively as a problem when these pigments are used as food coloring agents, a problem that must be countered by including an antioxidant in the food in question. Thus, they do not consider the peroxidase-mediated bleaching of these pigments to be of any practical use in itself.

Apart from this, it has been proposed to bleach pulp using peroxidases, see e.g. US Patent No. 4,690,895.

Peroxidases used for the purpose described here as detergent additives can be isolated from and can be produced by plants (eg horseradish peroxidase) or such micro-organisms as basidiomycetes, fungi, actinomycetes or bacteria. Some preferred microorganisms include strains of Fusarium, especially Fusarium oxysporum, strains of Streptomyces, especially Streptomyces thermoviolaceus or Streptomyces viridosporus, strains of Pseudomonas, preferably Pseudomonas putida or Pseudomonas fluorescens, strains of Coprinus, especially Coprinus cinereus, Coprinus macrorhizus or Coprinus cinereus f .microsporus, strains of Streptovercillium, especially Streptoverticillium verticillium ssp. verticillium, strains of Bacillus, especially Bacillus stearothermophilus, and strains of Coriolus, especially Coriolus versicolor, strains of Phanerochaete, especially Phanerochaete chrysosporium.

Specific examples of preferred strains are Coprinus cinereus f. microsporus IFO 8371, Coprinus cinereus IFO 30114, Pseudomonas fluorescens NRRL B-11, Streptoverticillium verticillium ssp. verticillium IFO 13864, Streptomyces thermoviolaceus CBS 278.66, Streptomyces viridosporus, ATCC 39115, Streptomyces badius ATCC 39117, Streptomyces phaeochromogenes NRRL B-3559, Pseudomonas pyrrocinia ATCC 15958, Fusarium oxysporum DSM 2 672 and Bacillus stearothermophilus ATCC 12978.

Other potential sources of useful peroxidases are listed in B.C. Saunders et al., op. ed., pp. 41-43.

Particularly preferred peroxidases are those which are active at the pH value that is typical for washing baths, that is at a pH value of 6.5-10.5, preferably 6.5-9.5, preferably 7.5- 9.5. More specifically, the peroxidases of interest for the present purpose are those which exhibit at least 25% of their optimal activity (that is, their activity at the pH optimum) in these pH ranges, e.g. as measured by the ABTS assay described in Example 1 below. Such peroxidases can be isolated by screening for peroxidase production using alkalophilic microorganisms, e.g. by the ABTS assay described in Example 1 below.

Other preferred peroxidases are those which exhibit good thermostability, especially those which retain at least 25% of their initial activity after 20 minutes at 40°C, as well as a good stability towards commonly used detergent components, e.g. nonionic, cationic or anionic surfactants, detergency builders, phosphate, etc., especially those which retain at least 25% of their initial activity after exposure to such detergent ingredients for 20 minutes.

Methods for producing enzymes to be used according to the invention are described in the field, see e.g. FEBS Letters 1625, 173(1), Applied and Environmental Microbiology, Feb. 1985, pp. 273-278, Applied Microbiol. Biotechnol. 26, pp. 357-360, Nature 326, 2 April 1987, FEBS Letters 4270, 209(2), p. 321.

Another group of useful peroxidases is made up of the ligninases: these enzymes which exert a strong peroxidase activity are instrumental in the breakdown of lignin, (e.g. in wood) and are produced by a variety of wood rot fungi. They have previously been proposed for pulp bleaching (see, for example, US Patent No. 4,690,895). Further useful peroxidases are halogen peroxidases, e.g. chlorine and bromine peroxidases, as they are able to oxidize halide ions to hypohalogenites which are powerful bleaching agents, apart from their ability to oxidize organic compounds.

The peroxidase can further be one that can be produced by a method comprising culturing a host cell transformed with a recombinant DNA vector carrying a DNA sequence encoding said peroxidase, as well as DNA sequences encoding functions that allow expression of the DNA sequence encoding the peroxidase, in a culture medium under conditions that allow expression of the peroxidase and to recover the peroxidase from the culture. The transformation of bacterial cells can be carried out according to conventional methods, e.g. as described in T. Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1982.

The selection of relevant DNA sequences and construction of vectors can also be carried out by standard methods, see T. Maniatis et al., op. one.

The medium used to grow the transformed host cells can be any conventional medium suitable for growing the host cells in question. The expressed peroxidase can conveniently be secreted into the culture medium and can be recovered therefrom by well-known methods including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components from the medium using a salt, e.g. ammonium sulfate, followed by chromatographic processes, e.g. ion exchange chromatography, affinity chromatography or the like.

Due to the possibility of reducing the amount of hydrogen peroxide required for bleaching stains on textiles by using a peroxidase as described above, it is possible to use an enzymatic process for hydrogen peroxide formation which previously had no technical interest in due to difficulties in generating sufficient quantities of hydrogen peroxide. Thus, the detergent additive according to the invention can additionally comprise an enzymatic system (that is, an enzyme and a substrate for this) which has the ability to generate hydrogen peroxide.

One such category of hydrogen peroxide-generating systems includes oxidases that have the ability to convert molecular oxygen and an organic or inorganic substrate into hydrogen peroxide and the oxidized substrate, respectively. In themselves, these enzymes are of limited or no use for bleaching stained textiles, as they produce too small amounts of hydrogen peroxide, but they can be used with great advantage in detergent additives according to the invention, as the presence of peroxidase ensures a efficient utilization of the hydrogen peroxide produced, as indicated above.

Preferred oxidases are those which act on cheap and readily available substrates which can be conveniently included in detergent mixtures. An example of such a substrate is glucose, which can be used for hydrogen peroxide production using glucose oxidase. Other suitable oxidases are urate oxidase, galactose oxidase, alcohol oxidases, amine oxidases, amino acid oxidase and cholesterol oxidase.

The detergent additive according to the invention can suitably be in the form of a non-dusting granule, a liquid, especially a stabilized liquid or a protected enzyme. Non-dusting granules can be produced e.g. as obvious in US patent no. 4,106,991 and 4,661,452 and may possibly be coated by methods known in the field. Liquid enzyme preparations can e.g. be stabilized by the addition of a polyol, e.g. propylene glycol, a sugar or a sugar a1coho1, lactic acid or boric acid according to established methods. Other enzyme stabilizers are well known in the art. Protected enzymes can be prepared according to the method disclosed in EP Patent No. 238,216.

The detergent additive can further include one or more other enzymes, e.g. protease, lipase or amylase, conventionally included in the detergent additive.

In another aspect, the invention relates to a detergent mixture comprising a surfactant and a detergent additive according to the first aspect of the invention, the enzyme exhibiting peroxidase activity being present in an amount corresponding to 0.1-100 mg peroxidase per liter wash bath.

Detergent mixtures according to the invention comprise, as mentioned, surfactants. These can be of anionic, nonionic, cationic, amphoteric or zwitterionic type, as well as mixtures of these surfactant classes. Typical examples of anionic surfactants are linear alkyl benzene sulphonates (LAS), α-olefin sulphonates (AOS), alcohol ethoxy sulphates (AES) and alkali metal salts of natural fatty acids.

Detergent mixtures according to the invention may contain other detergent ingredients known in the field, e.g. builders, anti-corrosion agents, sequestering agents, anti-fouling agents, perfumes, enzyme stabilizers, etc.

It is currently believed to be included that the detergent mixture according to the invention can contain the peroxidase in an amount corresponding to 0.1-100 mg of peroxidase per liter wash bath.

The detergent mixture according to the invention can be put together in any convenient form, e.g. as a powder or a liquid. The enzyme can be stabilized in a liquid detergent by incorporating enzyme stabilizers as indicated above. Liquid detergents can further include stabilized hydrogen peroxide precursors. Generally, the pH value of a solution of the detergent mixture according to the invention will be 7-12 and in some cases 7.0-10.5. Other detergent enzymes, e.g. proteases, lipases or amylases, can be included in the detergent mixtures according to the invention, either separately or in a combined additive as described above.

In a further aspect, the present invention relates to the use of the detergent additive according to the first aspect or the detergent mixture according to the next aspect for bleaching stains on textiles, which comprises treating textiles with a detergent additive or a detergent mixture as described above. In a bleaching process, textiles can be treated with an enzyme that exhibits peroxidase activity in the presence of hydrogen peroxide or a precursor thereof, possibly in the presence of an oxidase and a substrate for the oxidase. As indicated above, the process is particularly suitable for bleaching stains caused by naturally colored substances, e.g. polyphenols, such as found in fruit juice, wine, tea and the like. The bleaching treatment can conveniently be carried out in a soaking, washing or rinsing process. It is intended that the detergent additive or detergent mixture can also be

used for bleaching textiles during their production.

The present invention is further illustrated by the following examples.

Example 1

Cotton pads homogeneously soiled with unsweetened blackcurrant juice were subjected to a model washing treatment to assess the bleaching effect of the horseradish peroxidase + hydrogen peroxide system. At the level of 10 mmol per liter H2O2 (equivalent to approx. 1.6 g sodium perborate tetrahydrate per liter wash bath), a dosage of 5 mg/l horseradish peroxidase (Boehringer-Mannheim) was found to be beneficial.

Relative enzyme activity was measured before and after the washing experiment by a standard technique (described by R.E. Childs and W.G. Bardsley, Biochem. J. 145, 1975, pp. 93-103, which involved H 2 O 2 oxidation of ABTS at pH 7.5 , monitored at 418 nm (ABTS = 2,2<1->azino-bis(3-ethylbenzthiazoline-6-sulfonic acid, supplied by Boehringer-Mannheim as the diammonium salt). After washing, the activity was 70% of the initial activity.

Example 2

Cotton swabs homogeneously soiled with unsweetened blackcurrant juice, red wine and tea were subjected to a model washing treatment to assess the bleaching effect of the horseradish peroxidase/H 2 O 2 system.

The experimental conditions were:

Wash bath: 0.05 M sodium phosphate buffer with

pH=7.5 prepared with water of a hardness equivalent to

1.6 mM Ca<2+>.

Duration of washing: 60 minutes.

Temperature: 24°C.

Bleach: H2O2, dosed to 10 mM.

Enzyme: Sigma P-812 5 horseradish peroxidase

dosed to 2 mg/l.

Textile/bath ratio: 10 g/l.

Tap water was used to rinse the patches.

At 460 nM, the detergency values for treatment with enzyme + H 2 O 2 and treatment with only H 2 O 2 were found to be:

(The three types of rags were washed together and were accompanied by unstained cotton rags. The last line in the table relates to the unstained rags).

Similar experiments were performed at pH 8.3 and at both pH values in the presence of an anionic surfactant (linear alkyl benzene sulfonate) and a nonionic surfactant. None of these changes significantly influenced the bleaching effect, and the activity of the enzyme in the washing solution, monitored by the ABTS assay described in Example 1, was essentially the same in all experiments (a relative activity of +/- 30%).

Example 3

Cotton rags homogeneously soiled with beetroot juice were subjected to several model washing treatments to assess the bleaching effect of the system H 2 O 2 + peroxidase derived from Coprinus macrorhizus + H 2 O 2 .

The experimental conditions were:

Wash bath: Potassium phosphate buffer 0.05 M

(see below) with pH adjusted to 7.0.

Washing duration: Varies, as specified

below.

Temperature: 24°C.

Textile/bath ratio: 11 g/l.

Hydrogen peroxide: 0.2 mM.

Enzyme: 20 mg/l peroxidase extracted from Coprinus macrorhizus (Chemical Dynamics Corporation, New Jersey, U.S., prod. no. 70-9590-00).

Assessment of

bleaching effect: determined as described in example 2.

The results were as follows:

In experiment (1), washing was followed by soaking overnight. In experiments (2) and (3) the buffer solution was made with water with a hardness equivalent to 1.6 nM Ca<2+>, in (1) and (4) the buffer was made with demineralised water.

Claims (9)

1. Detergent additive for bleaching stains on textiles, characterized in that it comprises (a) an enzyme that exhibits peroxidase activity, where the enzyme is in the form of a non-dusting granule, a liquid, in particular a stabilized liquid, or a protected enzyme, and (b) hydrogen peroxide, a precursor of hydrogen peroxide or an enzymatic system capable of generating hydrogen peroxide. 2. Detergent additive as stated in claim 1, characterized in that the peroxidase is one that can be produced by a microorganism, e.g. a bacterium, fungus, actinomycetes or basidiomycetes. 3. Detergent additive as stated in claim 1, characterized in that the peroxidase is of plant origin. 4. Detergent additive as stated in any of claims 1-3, characterized in that the peroxidase is active at pH 6.5-10.5, preferably 6.5-9.5, preferably 7.5-9.5 . 5. Detergent additive as stated in claim 1, characterized in that the precursor to hydrogen peroxide is a perborate or a percarbonate. 6. Detergent mixture, characterized in that it comprises a surfactant and a detergent additive according to any one of claims 1-5, the enzyme exhibiting peroxidase activity being present in an amount corresponding to 0.1-100 mg peroxidase per liter wash bath. 7. Detergent mixture as stated in claim 6, characterized in that the precursor for hydrogen peroxide is a perborate or a percarbonate. 8. Use of a detergent additive according to any one of claims 1-5, or a detergent mixture according to claim 6 or 7, for bleaching stains on textiles. 9. Application according to claim 8 where the bleaching is carried out in a soaking, washing or rinsing process.