WO1997000834A1

WO1997000834A1 - Enzymatic method for the oxidative breakdown of fluorescent compounds

Info

Publication number: WO1997000834A1
Application number: PCT/EP1996/002512
Authority: WO
Inventors: Philippe Schyns; Jürgen REINERS; Joachim König; Ulrich Feldhues; Rainhard Koch; Eckhard Wenderoth; Udo Eckstein; Uwe Vogt
Original assignee: Bayer Aktiengesellschaft
Priority date: 1995-06-23
Filing date: 1996-06-10
Publication date: 1997-01-09
Also published as: DE19523389A1; EP0835225A1

Abstract

Fluorescent compounds in aqueous systems can be broken down by treatment of said aqueous systems with an enzyme from the group comprising the oxidases and peroxidases and with an oxidising agent taken from the group of compounds containing or forming O2, H2O2 and O2, or H2O2, at a temperature of between 5 and 90 °C and a pH value of 5-10. The treatment can be carried out in the presence of surface-active auxiliary agents.

Description

Enzymatic method for the oxidative degradation of fluorescent compounds

The invention relates to a method for the enzymatic degradation of fluorescent compounds which are located in aqueous systems, in which such aqueous systems with one or more oxidases or peroxidases and O ₂ , H, O ₂ or compounds which contain O ₂ or Form H ₂ O ₂ are treated.

Fluorescent compounds are mainly used as optical brighteners. These are substances that in solution or on a substrate UV light, e.g. absorb the proportion of daylight at approximately 280 to 430 nm and emit the absorbed energy as blue fluorescent light of the wavelength of approximately 400 to 500 nm. Textiles, paper, cardboard or cardboard appear whiter and lighter after treatment with optical brighteners because part of the area of the daylight spectrum that is not perceptible to the eye is visible

Light is converted. Brighteners absorb in the UV range or reduce the reflection in this range. At the visible wavelengths, usually with a maximum at 430 to 450 nm, they increase the reflection due to the emitted fluorescence. Brighteners are the more effective the whiter the substrate is. However, they are not a substitute for bleach. The industrially important ones

Brighteners are compounds which are derived from 4,4'-diaminostilbene-disulfonic acid, distyryl-benzene, distyryl-biphenyl, stilbenyl-2H-triazole, certain benzoxazoles, benzofurans and coumarins.

Optical brighteners are used in a variety of ways. As is well known, detergents brighteners are known to be added in order to replace the brightener components present in textiles which, owing to the poor fixation to the fibers, can be eluted when washing *. When such washing processes are carried out industrially, discontinuous or continuous processes analogous to the known dyeing processes are used.

Optical brighteners are also used in the production of uncoated and coated papers, in particular printing papers. The degree of whiteness can be increased considerably by using very small amounts; Brighteners work with the greatest efficiency in the concentration range of 0.002 to 2.0% by weight, based on the substrate, and should be distributed as monomolecularly as possible on the substrate. For paper production, brighteners can be added directly to the pulp suspension from which the paper is formed by dewatering. The most common application in practice, however, is the use in the paper surface, the brightener being applied to the paper surface together with other auxiliaries, such as surface sizing agents or starch, for example using a size press. Another important area of application is the addition of brighteners to coating colors. In the paper industry, inexpensive triazinyl-aminostilbenes or di-styryl-biphenyls are preferably used.

For a long time, environmental and safety aspects have been given high consideration in the development, manufacture and use of optical brighteners, for example in liquid or powder formulations. For example, anionic brighteners, especially those with sulfonic acid groups, have been developed so that they behave in relation to a cellulose noun and are fixed against bleeding.

For use with food packaging paper, it is required that the brightener does not migrate to the food, i.e. that grade 5 is achieved in the blood test according to DIN 53.991, sheet 2. This applies analogously to hygiene paper (DIN 53.991, sheet 4).

For some time now, increased bleeding from optical brighteners or other fluorescent compounds has been observed increasingly in the case of paper and cardboard which are not or only slightly brightened; this is obviously related to the increased use of secondary fibers, the cellulosic structure of which is damaged and therefore no longer contains any optical brighteners originally present. This represents particularly for manufacturers of

Food packaging paper poses a problem, since in this area the approval is required to pass the grade 5 blood test. (Scale from 1 to 5; higher values indicate greater resistance to bleeding; method DIN 53.991, sheets 2 and 4). In addition to the above-mentioned recycling of lightened papers, the replacement of chlorine bleach in cellulose production by TCF bleach (TCF = Totally Chlorine Free) is suspected as a possible cause. The influence of deinking chemicals on the maintenance of fixation of brighteners is still largely unknown. The behavior of optical brighteners during recycling has also not yet been extensively investigated. In the area of Waste paper recycling would have to be achieved, for example, that the brightener remains firmly attached to the fiber or that it is fixed from the outset in a suitable manner in order to prevent bleeding. This could ensure that originally lightened secondary fibers could also be used as raw materials for papers that were not subsequently lightened, without having any adverse effect. If fluorescent compounds from the pulp production or brighteners from the secondary fiber materials are only insufficiently fixed in the paper due to mechanical or chemical modification, the use of certain types of paper for packaging food is excluded, since in this case the bleeding test is no longer passed .

In addition to the fact that brighteners remain in the paper and the resultant discharge of these brighteners from the production cycle of a paper mill, there is also the possibility of removing the brightener from the fiber after it has been desorbed by treating the washing water or the flotates from the circulation of the

To remove paper pulp. However, due to the current conditions in practice and the use of different chemicals in different concentrations, it is hardly possible to control the whereabouts and the breakdown of brighteners. Optical brighteners were previously destroyed by hypochlorite in the processing of waste paper. However, this produces chlorine-organic compounds which, in addition to the unfavorable toxicological assessment, in any case increase the AOX content in the waste water. Another method of destroying optical brighteners is oxidation by persulfate in a strongly alkaline medium. Disadvantages of this treatment method are a high chemical requirement and the pollution of the environment by remaining salts.

It is also possible to control the fluorescence of the brighteners using cationic components such as e.g. To extinguish polyamide amines, polyamide amine-epichlorohydrin resins, dicyandiamide condensates or other fluorescence quenchers. Here, the fluorescent compound is not destroyed, but only rendered ineffective by complex formation.

Further problematic wastewater is generated in the private or commercial laundry of textiles, since the detergents used, as already mentioned above, often contain optical brighteners, which are more washed out to supplement them Brighteners are intended to serve in the textile, but not all of them remain in the textile as intended, but rather appear in the washing machine's waste water.

Brighteners that are contained in wastewater from private households or commercial plants, especially from the paper, textile and detergent industries, are difficult to break down in biological wastewater treatment processes. They are largely adsorbed on sewage sludge, which in turn has to be disposed of at great expense.

In addition to the above-mentioned problems with the recycling of waste paper and with various other types of waste water, a problem can also arise in the manufacture of lightened paper types and cardboard boxes in that the compulsion to avoid waste and the careful use of resources such as fibers, auxiliaries and water, would like to keep the water cycles required here closed, but must expect an enrichment of the brightener, since a complete pulling up of the brightener on the cellulose fibers is not guaranteed. A further increase in the amount of brightener in the water cycle can result from the use of a lightened committee.

Removal of lightening residues from the white water of the paper machines is not possible with the methods currently available. The problem of the enrichment of optical brighteners in the water circuits of the paper industry is exacerbated by the rapidly increasing demand for lightened papers with additional demands on the degree of whiteness.

There is therefore a great need for an environmentally friendly method for removing optical brighteners as the most important representatives of fluorescent compounds, the removal being carried out in the wastewater treatment (wastewater from various sources, as explained above), in the control and

Monitoring of closed circuits and for various uses of optical brighteners in the paper, textile and detergent industries. The problem is particularly important for the paper industry against the background of rapidly increasing waste paper recycling quotas, which are currently around 30 to 50% in Europe.

The object of the invention is therefore to provide a method which degrades fluorescent compounds in such a way that subsequent biological wastewater treatment action is possible. The method according to the invention should also be applicable to TCF pulps in which only an oxidative removal of fluorescent compounds, for example by chlorine or hypochlorite, has hitherto existed. Furthermore, the disadvantages of the known methods with their high use of chemicals and the formation of chlorine-containing compounds are to be overcome; In the case of persulfate degradation, for example, high pH values have to be set, which in turn leads to a high salt load after the necessary neutralization. In addition to the chemical and toxicological disadvantages mentioned, the use of the chemicals mentioned is cost-intensive. Such processes are therefore in need of economic improvement.

It has been found that oxidases and peroxidases are suitable for degrading fluorescent components, for example optical brighteners in waste water and circuits, which contain such fluorescent compounds, using O- or H ₂ O ₂ .

Some applications of peroxidases have already been proposed: it is known from WO 89/09813 that peroxidases can be used for the oxidation of phenolic bodies. Furthermore, peroxidases for bleaching wood pulp, for treating wastewater from the pulp industry or for use in textile washing are proposed, in the latter case in particular to prevent back training. EP 406 617 proposes ligninases in combination with xylanases for the bleaching of paper pulp. Enzymes for the degradation of lignin are claimed in EP 429 422. According to US Pat. No. 5,370,770, soybean peroxidase is used for deinking waste paper. The degradation of phenolic impurities with the aid of peroxidases is also proposed in US Pat. No. 5,178,762.

The use of oxidases and peroxidases for enzyme-catalyzed oxidative

However, degradation of fluorescent compounds, for example optical brighteners, has obviously not been successful to date, at least not described, although the problems mentioned above are urgent and known to those skilled in the art.

The invention relates to a method for the oxidative degradation of in aqueous

Fluorescent compounds present in systems, which is characterized in that such aqueous systems with at least one enzyme from the group of oxidases and peroxidases, which are both present as such and can also be associated with at least one further enzyme with hydrolytic activity, and with an oxidizing agent from the group of mixtures containing or forming O ₂ , H, O ₂ and O ₂ or H ₂ O ₂ at a temperature of 5 to 90 ° C and a pH of 4 to 10 treated.

Treatment can be in the absence or in the presence of surfactants

Aids are carried out.

Aqueous systems in the sense of the invention are waste water from the production or treatment of textiles, fiber structures, such as paper, cardboard or cardboard, the fibers and polymers on which they are based, in particular those based on cellulose, and also aqueous dispersions or suspensions of such textile fiber structures, fibers and polymers. The aqueous system according to the invention can also be a treatment liquor, for example a textile treatment liquor, preferably one for clothing textiles. Fiber structures, such as paper, cardboard or cardboard, in particular lightened, coated or uncoated printing and writing papers, are usually opened in a pulper when they are recycled, freed of coarse impurities and then subjected to washing or flotation deinking. The enzymatic treatment according to the invention for the degradation of optical brighteners can take place before, during or after the detachment of the printing ink components. The pulp cleaned in this way can then be subjected to a further bleaching step, likewise

Enzymes can be used. In the event that the optical brightener is absorbed on the fiber, but is to be used in the course of recycling for the production of unlightened paper, cardboard or cardboard, it may be advantageous to use further enzymes in addition to the oxidases or peroxidases, for example the desorption of the optical brightener from the fiber.

The method according to the invention for oxidative degradation with the aid of enzymes from the groups of oxidases and peroxidases is in principle accessible to all fluorescent compounds, in particular those which are used as optical brighteners. A large part of such fluorescent compounds contains, as already mentioned above, the 4,4'-diaminostilbene disulfonic acid, the distyryl-benzene, the distyryl-biphenyl, the stilbenyl-2H-triazole, the benzoxazole, the benzofuran or the coumarin as the molecular framework . Of these fluorescent compounds, those are particularly important which contain the 4,4'-diaminostilbene disulfonic acid or the distyryl biphenyl as the molecular backbone. Especially fluorescent compounds which contain the 4,4'-diaminostilbene-disulfonic acid skeleton can preferably be subjected to the method according to the invention. Important examples of fluorescent compounds are, for example, those of the formula

wonn

X, amino, methylamino, ethylamino, dimethylamino, diethylamino, 2-hydroxyethyl amino, 3-hydroxypropylamino, di- (2-hydroxyethyl) amino, di- (2-hydroxypropyl) amino, 2 -Sulfo-ethylamino, morpholino, anilino, chloranilino, sulfoanilino, methylanilino or disulfoanilino and

X _{2 is} hydroxy, methoxy, ethoxy, methoxyethoxy, chlorine or X,

is a hydrogen, alkali metal, amine or ammonium ion,

as well as those of the formula

wherein

X ₃ and X ₄ denote hydrogen, methyl, ethyl, phenyl or sulfophenyl,

Z has the meaning given above,

as well as the formula V ^XX (HI),

wonn

X _{5 is} hydrogen, methyl, ethyl, methoxy, ethoxy, chlorine or sulfo, in all cases

Z is hydrogen, an alkali metal, amine or ammonium ion.

Such fluorescent compounds are known, for example, from EP 409 028. Important individual examples of this are those of the following formulas (A) to (G), some of which are products from Bayer AG under the trade name Blankophor:

^{^} ^

(A)

> ^it

(B)

^{> Q}

(C)

(D)

(E)

(F)

A M ^

(G)

Enzymes from the group of oxidases and peroxidases are suitable for use in the method according to the invention, which according to "Enzyme Nomenclature 1984, Academic Press Inc., New York, London "(EC) belong to the larger group of oxidoreductases that catalyze the electron transfer between different substances. In the following, examples of such enzymes are mentioned, which are identified in the EC by combinations of numbers and can be found there in this way:

Peroxidases (E.C. 1.1 1.1.7) are enzymes which catalyze the oxidation of a substrate with hydrogen peroxide and which are obtainable from plant, microbial or animal sources. Examples include: horseradish peroxidase, soybean peroxidase or a peroxidase from Coprinus. for example Coprinus cinereus or Coprinus macrorhizus. or Bacillus. for example

Bacillus pumilus or Bacillus myxococcus. Trametes. Rhizoctonia. Pseudomonas. Streptomyces candida. Curvularia. Cercospora. Polyporus pinsitus. Lignin peroxidase, Mn-dependent peroxidases, peroxidases from Phanerochaete. Basidiomycetes. The peroxidases from WO 93/24618 with improved stability, chloroperoxidase and lactoperoxidase may also be mentioned.

Oxidases (E.C. 1.10.3.1)

Cellobiose oxidase from Phanerochaete chrysosporium or Humicola. Glucose oxidase, catechol oxidase, polyphenol oxidase. The oxidases also include laccases (E.C. 1.10.3.2), in particular laccases from Trametes versicolor. Phanerochaete chrysosporium; such enzymes are for example from

Polyporus pinsitus (Trametes villosa) produces.

The peroxidases are used together with H ₂ 0 ₂ or a compound containing or forming H ₂ O ₂ as the oxidizing agent. In addition to the H ₂ O, itself, usually in the form of differently highly concentrated and commercially available aqueous H ₂ O, solutions, the following should therefore be mentioned: perborates, persulfates, percarbonates,

Peresters, peroxides, Fenton's reagent, peracids or enzymatic systems which form H ₂ 0 ₂ , such as glucose / glucose oxidase / oxygen, amino acid oxidase, urea oxidase, cholesterol oxidase, amine oxidase or alcohol oxidase. Oxidases, including laccases, are used with 0 ₂ or O ₂ containing or forming compounds or 0 -, - sources.

In addition to the enzymes and oxidizing agents mentioned, it may be advantageous to use other auxiliaries during the treatment, such as metal salts (for example alkali metal halides, sulfates, phosphates, acetates etc.), saccharides (for example Cellobiose, glucose, fructose, ribose, mannose, galactose, arabinose, trehalose, xanthans), halide ions, buffer solutions (e.g. phosphate, borate, acetate buffer) and reducing or oxidizing agents (e.g. NaHSO ₃ , Na ₂ S ₂ O ₄ or formamidine - sulfinic acid or perborate, periodate or percarbonate). These aids can be suitable for controlling the electrochemical potential of the mixture.

It can also be advantageous to use accelerators for the action of the peroxidases or oxidases. The effect of such accelerators is attributed to the fact that short-lived radicals or other oxidized states are formed in the reaction system, which can contribute to the oxidative degradation of the fluorescent compounds. Such accelerators are, for example, azines of the formula A = NN = B (WO 94/12620), and also N-methylphenothiazine, 3,3 ', 5,5'-tetramethylbenzidine, 4-amino-4'-methoxy- stilbene, 2,2'-azino-bis- (3-ethylbenzo-thiazoline-6-sulfonate). Metal ions, halide ions, phenol, p-hydroxybenzoic acid, p-hydroxybenzenesulfonate, p-hydroxycinnamic acid, 7-hydroxycoumarin, guaiacol or vanillin can also be used as accelerators. The accelerator can e.g. in an aqueous system with the enzyme in a concentration of 0.01 to 100 μM, preferably 0.1 to 50 μM, particularly preferably 1 to 10 μM.

The enzymes mentioned from the group of oxidases and peroxidases can either be present as such alone or be associated with at least one further enzyme with hydrolytic activity. The latter case has the advantage that the enzyme preparations obtained from the sources mentioned above (plants, microorganisms or fungi), which are often a mixture of several enzymes, do not have to be separated into individual enzymes. This avoids additional costs. Such further enzymes with which oxidases and peroxidases can be associated are, for example, hydrolases, such as cellulases, for example from Phanerochaete. Humicola. Fusarium. Pseudomonas. Trichoderma reesei or Aspergillus: hemicellulases, preferably xylanases, such as Pulpzyme HC ^® ; Lipases, for example from Pseudomonas. Humicola. Candida, Chromobacter. Aspergillus: (. Fa products of Novo Nordisk) proteases, such as MPX NUE 0.6, Aquaderm ^®, Pyrase ^®, Alcalase ^®, Esperase ^®; Amylases as Denimax ^®, Termamyl ^® and others; Pectinases, dehalogenases and pullulanases. Such further enzymes are mentioned, for example, in WO 91/17243, WO 91/17244, WO 91/10732, US 5,338,403, WO 94/23053 and WO 91/14019. It can furthermore be advantageous to mobilize fluorescent compounds, for example brighteners, which are adsorbed on the fiber, by also using surface-active aids. Anionic, cationic or nonionic and amphoteric surfactants are suitable as such surface-active compounds. Examples of these are salts of fatty acids, ammonium salts of fatty amines, polyethers with long-chain alkyl radicals, the polyethers being formed by ethoxylation and / or propoxylation, partial fatty acid esters of glycerol, trimethylolpropane, pentaerythritol, oleic acid sarcosides, sulfosuccinates, polyacrylic acid derivatives with hydrophobic side chains or End groups, polyamide amine emulsifiers, etc. Such surfactants are

Expert known for a long time.

The method according to the invention is carried out at a temperature of 5 to 90 ° C., preferably 10 to 60 ° C., particularly preferably 20 to 55 ° C. and in the pH range of 4 to 10, preferably 5 to 8. The residence time of the aqueous system to be treated in the context of the method according to the invention depends on many factors, for example on the concentration of the fluorescent compound, the concentration of the enzyme, the concentration of the oxidizing agent, the temperature and the pH. In many cases, a time period of at most 200 minutes, in many cases of at most 60 minutes and in some cases of at most 10 minutes, will be required in order to recognize from the samples that the fluorescence has largely been quenched or has been quenched to an extent that is striven for in individual cases. The quenching of the fluorescence on the basis of a sample taken here indicates that the fluorescent compound to be oxidized has been oxidatively split into at least two fragments. This can be proven by chromatographic methods such as TLC or HPLC.

To carry out the method according to the invention, a solution of an oxidase or peroxidase and one of the oxidizing agents mentioned are added to the aqueous system. The concentration of the fluorescent compound can be in the range from 3 parts per billion (ppb) up to 1% by weight or more if, for example, it is a concentrated medium with a strongly fluorescent compound.

The amounts of enzyme used depend on the content of fluorescent compound in the aqueous system. In general, the peroxidase is in a Amount of 1 to IO ⁷ PODU (= peroxidase units) per 1 g of the fluorescent compound used. The oxidase is used in an amount of 1 to 10 ⁷ laccase units per 1 g of the fluorescent compound. The term 1 PODU is to be understood as the amount of enzyme which, under standard conditions (0.1 M phosphate buffer, pH = 7, temperature = 30 ° C., 0.88 mM H ₂ O ₂ , 1.67 mM

ABTS) in 1 minute the reaction of 1 μmol H ₂ O _{2 was} catalyzed in a system in which ABTS (= 2,2'-azino-bis- (3-ethyl-benzothiazoline-6-sulfonic acid)) was oxidized. The term 1 laccase unit is to be understood as the amount of enzyme which under standard conditions (0.1 M sodium acetate buffer, pH = 5, temperature = 30 ° C., oxygen) catalyzes the conversion of 1 μmol ABTS per minute. Under these standard conditions, a green-blue dye is produced, which can be measured photometrically at 418 nm.

Assuming a usual amount of 1% by weight of brightener on absolutely dry (dry) paper, the enzyme dosage required to disappear the fluorescence is, for example, 100 to 10 ⁸ units per kg of dry paper, preferably 100 to 10 ⁶ units per kg of dry paper, particularly preferably 100 to 10 ^ units / kg of dry paper. This corresponds to an amount of 10 μl to 10 l of enzyme solution per kg of dry paper, preferably 10 μl to 100 ml per kg of dry paper, in particular 10 μl to 10 ml / kg dry paper, if an activity of 10,000 units is used for an enzyme solution / g as the basis. Deviating

Enzyme activity can be compensated for by making corrections in the dosage.

The H ₂ O ₂ concentration in the aqueous system to be treated is generally 0.001 mmol to 2.0 mol, preferably 0.01 mmol to 1 mol, per liter. Based on the dry substance that is present in the aqueous system to be treated, preferably 0.001 to 5% by weight of H ₂ O _{2 is} sufficient. If one wishes to focus on the amount of fluorescent compound, the R, O -, - amount is preferably 1 x 10 ^"5 to 1 x IO ^{" 2} % by weight, based on the fluorescent compound. If O _{2 is used} as the oxidizing agent, pure oxygen can be used, but atmospheric air or oxygen-enriched atmospheric air can also be used; Since the O ₂ concentration of the atmospheric air is normally sufficient in laccase systems, this option is also used. With a contact time of the aqueous system of 0.1 to 100 minutes at pH = 7 and 37 ° C., no fluorescent compound, recognizable by its fluorescence, is more detectable. The reaction time can be shifted to shorter values by correspondingly increasing the amount of enzyme.

If the aqueous system to be treated is a suspension or slurry which accordingly has a solids content, this solids content is 0.1 to 30% by weight, preferably 0.5 to 10% by weight, of the total suspension or slurry.

In the cases where the fluorescent compounds are in the adsorbed state on a solid surface, it is advantageous to bring the fluorescent compound into solution beforehand. Known anionic surfactants or, in the case of cellulose, cellulases or in particular are suitable for this purpose

Endoglucanases.

The course and completeness of the oxidative degradation of the fluorescent compound can be followed by measuring the fluorescence. For this purpose, a sample of the aqueous system (waste water or dispersion or aqueous slurry) can be introduced directly into a measuring device; however, chromatographic methods can also be used to separate the substances present in the aqueous system. After the reaction has ended, the enzyme can be deactivated by simply changing the pH and / or increasing the temperature. In closed circuits, this is generally not necessary, so that further enzyme need only be added if the enzyme activity decreases. An addition of catalases can also serve to interrupt the enzyme activity. In a further embodiment, use can also be made of the immobilization of enzymes known to the person skilled in the art, a carrier material with enzymes in immobilized form being arranged, for example, in a column and brought into contact with the aqueous system to be treated; Such an embodiment is of course only applicable to dissolved fluorescent compounds and not to dispersions or slurries.

The method according to the invention generally requires a significantly lower use of oxidizing agents and auxiliaries and enables faster and more environmentally friendly disposal of fluorescent compounds. In comparison, the chemical requirement for a non-enzyme-catalyzed degradation with potassium perox odi sulfate at pH = 10.5 and 60 ° C, a consistency of 5% by weight of a dispersion and a treatment time of 180 minutes 1 to 2% by weight of persulfate, based on the solid (for example cellulose). In the case of treatment with chlorine solution at pH = 3, it is necessary to use 0.5% by weight of active chlorine, based on solids (for example cellulose), in order to quench the fluorescence after 60 minutes at a consistency of 2.5% by weight to reach. When using cationic polymeric auxiliaries, up to 2% by weight of the auxiliary is used in order to achieve quantitative quenching when 0.8% by weight of fluorescent compound is used. Dismantling does not take place here, or only to a minor extent.

The fragments obtained by the enzymatic degradation of the fluorescent compounds are generally more accessible to microbiological degradation in a wastewater treatment plant than the untreated fluorescent compounds.

All percentages relate to percent by weight.

example 1

A stock solution with a concentration of 0.05% (a 25% solution) of a brightener of the formula (A) (Example 12 of EP 0 409 028), see formula listing above, was in a 50 mM potassium phosphate buffer ( pH 7). The sample was incubated with 500 units of a horseradish peroxidase (Fluka, No. 77333) at 28 ° C. The enzyme had an activity of 700 units / mg. 1 unit oxidizes 1 μmol ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) per minute at 25 ° C and pH 6.

The concentration of the peroxidase was 100 units / μl brightener (calculated for 25% delivery form). The hydrogen peroxide concentration was 0.15 mmol / 1. After various times, samples were taken from each test batch, centrifuged and their absorption and fluorescence examined.

The absorption spectrum was recorded with a spectrophotometer in the wavelength range between 200 and 500 nm. Measurement of emission at

440 nm was carried out using a fluorescence spectrophotometer at an excitation wavelength of 280 or 350 nm.

Conditions of thin-layer chromatographic separation: In addition, the test solutions were examined using RP-TLC (Reversed-Phase Thin-Layer Chromatography):

0.25 mm silica gel (stationary phase with fluorescence indicator, RP 18 W / UV 254,

Macherey-Nagel),

Aqueous eluent containing 8.75% (w / v) oxalic acid and 0.14% (w / v)

Sodium heptanesulfonate / methanol / acetonitrile (4: 1: 1) in water.

Results:

During the enzyme treatment (21 hours) the intensity of the absorption band at 350 nm decreased by 72%. After the enzyme treatment, only a small emission was observed at 440 nm, which occurs in an intact brightener molecule by excitation with a wavelength of 280 or 350 nm. The fluorescence was weakened by 90 to 95%. The stilbene derivative, which elutes as a spot at R _f = 0.83, was converted by enzyme treatment into three new spots which eluted at the Rf values 0.875 / 0.765 / 0.73. After less than 200 minutes the spot of the educt was no longer visible.

Example 2

A stock solution with a concentration of 0.05% (a 25% solution, v / v) of a brightener of the formula (A) was prepared in a 50 mM potassium phosphate buffer (pH 7). The sample was incubated with 10 units of a peroxidase (Novozym 502, Novo Nordisk) at 37 ° C. The enzyme is a recombinant heme-containing enzyme that was originally isolated from Basidiomycetes and has an activity of 10,000 PODU / g. 1 PODU oxidizes 1 μmol ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), c = 1.67 mM per minute at 30 ° C, pH 7 and an H ₂ O ₂ concentration of 0 , 88 mM. The enzyme also has a side activity as an amylase.

The concentration of the peroxidase was 7 units / μl brightener (calculated for

25% form of delivery). The hydrogen peroxide concentration (use: 30%) was 0.15 mmol / 1. After various times, samples were taken from each test batch, centrifuged and their absorption and fluorescence were examined as in Example 1.

Results:

The enzyme treatment led to a reduction of the absorption at 350 nm by approx. 90%. The emission at 440 nm, which is measured when excited with a wavelength of 280 nm or 350 nm, was attenuated by 95 to 98%.

The stilbene derivative, which eluted without an enzyme treatment (100 minutes) as a spot at R ^ = 0.83, was cleaved by enzyme treatment so that three new ones

Spots at the R _f values of 0.875 / 0.765 / 0.73 eluted. After 100 minutes, the spot of the educt was no longer visible. After 100 minutes, the brightener was completely fragmented. Example 3

Example 1 was repeated with the change that peroxidase from Example 2 was used in a concentration of 700 units per μl of brightener. After a few seconds the brightener was completely fragmented.

Example 4 = control test

A corresponding experiment analogous to Example 1, in which only hydrogen peroxide but no enzyme was used, did not lead to a decrease in fluorescence under identical conditions. The control by means of thin layer chromatography showed only one spot which could be assigned to the brightener.

Example 5 (application example

100 g base paper, consisting of 70% bleached pine sulfate and 30% bleached birch sulfate pulp, which contained 0.25% active substance according to formula (A) above and had a CIE whiteness of 125, was obtained at a consistency of 5% opened (2000 ml, 3000 rpm).

First, 7000 Cellusoft L endoglucanase units (EGU) were added

(10 h, 50 ° C).

At 37 ° C. and pH 6, 30,000 PODU (3 ml), which were determined according to Example 2, were added to the peroxidase from Example 2. Then hydrogen peroxide (concentration in the stock suspension = 0.15 mol / liter) was added and kept at 37 ° C. for 120 minutes. The isolated pulp was partially broken down.

After the enzyme treatment had ended, the fiber pulp was diluted to a consistency of 0.5% and a paper sheet was formed on a Rapid-Köthen sheet former. The paper sheet formed has a CIE whiteness of 85. An unlightened paper usually has a whiteness of 80. The above value of 85 therefore indicates the breakdown of the brightener. Example 6 = comparative example

The brightener quenching can also be achieved by treating a 0.5% solution of brightener of the formula (A) at pH 10.5 / 60 ° C. with 2% potassium peroxodisulfate for 120 minutes. However, the conditions for the breakdown of the brightener are much more drastic (pH, temperature) and require a higher one

Amount of oxidizing agent used with a longer dwell time.

Claims

1. Method for the oxidative degradation of fluorescent compounds in aqueous systems, characterized in that such aqueous systems with at least one enzyme from the group of oxidases and peroxidases, which are present as such alone and with at least one further enzyme with hydrolytic Activity can be associated, and with an oxidizing agent from the group of O ₂ , H ₂ O ₂ and O ₂ or H ₂ O ₂ containing or forming compounds at a temperature of 5 to 90 ° C and a pH of 4 to 10 treated.

2. The method as claimed in claim 1, characterized in that the enzyme with hydrolytic activity that is suitable for the oxidation of the oxidases and peroxidases is at least one of the enzymes which break down cellulose or hemicellulose, the lipases, amylases, proteases and dehalogenases.

3. Method according to claims 1 and 2, characterized in that the associated enzyme with hydrolytic, cellulose or hemicellulose degrading activity is cellulase or xylanase or a mixture of both.

4. The method according to claim 1, characterized in that the treatment is carried out at 10 to 60 ° C, preferably 20 to 55 ° C.

5. The method according to claim 1, characterized in that the treatment is carried out at a pH of 5 to 8.

6. The method according to claim 1, characterized in that the aqueous system to be treated is a waste water or a circulating water of the treatment or the production of cellulosic material or a suspension of cellulosic material.

7. The method according to claim 6, characterized in that the cellulosic material is paper, cardboard or cardboard.

8. The method according to claim 1, characterized in that the fluorescent compounds to be degraded as a molecular structure, the 4,4'-diaminostilbene disulfonic acid, the distyryl-benzene, the distyryl-biphenyl, the stilbenyl-2H-triazole, the benzoxazole, the benzofuran or the coumarin.

9. The method according to claim 8, characterized in that the fluorescent compounds to be degraded contain the 4,4'-diaminostilbene disulfonic acid or the distyryl biphenyl, preferably the 4,4'-diamino stilbene disulfonic acid, as the molecular structure.

10. The method according to claim 1, characterized in that the treatment is carried out in the presence of surfactants.