DE19804583A1 - Multi-component system for changing, breaking down or bleaching lignin, lignin-containing materials and processes for its use - Google Patents

Description

The present invention relates to a multi-component system for Changing, breaking down or bleaching lignin, lignin-containing mate rialien and methods for its application.

The main goal of all processes for the production of pulp is the removal of the lignin from the vegetable raw material. Typically, this goal is achieved by using appropriate oxidative or reductive chemical process steps who applied those who ultimately modify the lignin in a way in which it can be easily extracted from the pulp product leaves. Traditionally, in the chemical process step chlorine or chemicals containing chlorine, such as hypochlorite or Chlorine dioxide.

More recently, instead of this or in addition to it chlorine-free compounds are used. These include e.g. B. oxygen, hydrogen peroxide, peroxyacids or ozone. Typically, the pulp to be processed is between between the different chemical treatments tion step subjected to which alkali, e.g. B. NaOH contains.

As an additional chlorine-free process step in the chemical Lignin removal processes are enzymatic processes known to do the chemical removal of lignin in a lot stage delignification and bleaching sequence should facilitate. This process step can be single-stage or multi-stage in se quantitative combination with chemical process steps are performed (WO 91/11553). As advantages of this procedure steps are the improvement of the achievable whiteness or  the reduction of the need for chemicals, especially the ones used Amount of chlorine.

EP 0 487 557 also discloses chemical delignification process using oxygen, additional individual enzymes me or mixtures of enzymes from related classes Zen. These are e.g. B. enzyme preparations, which different Hemi contain cellulases.

From US 5,374,555 is a chemical process with additional enzymatic pulp treatment using enzymes known from the group of proteases. Here too, the treatment tion carried out as a separate process step, which in sequential combination with chemical process steps Advantages leads.

WO 91/11552 describes the use of oxidizing and hydro lytic enzymes in a mechanical manufacturing process of wood pulp. Additives such as B. ascorbic acid to do this serve to set the redox potential to values <200 mV.

In addition to the described enzymatic processes, which additionally Lich to chemical processes for the treatment of the pulp are also enzymatic processes for the treatment of Described pulp.

Such processes use phenol oxidizing enzymes. examples for such enzymes are enzymes from the group of peroxidases (E.C. 1.11.1) and oxidases (E.C. 1.10.3). The enzymes use either Hydrogen peroxide or molecular oxygen as an electron accept gate. The use of these oxidative enzymes in the bleaching is wrote (e.g. Paice, M.G., 1995). Find use z. B. Man gan peroxidase (MnP), lignin peroxidase (LiP) or laccase (E.C. 1.10.3.2). While LiP and MnP are typically used as cosubstrates  Need hydrogen peroxide, laccase works with oxygen as Electron acceptor.

The successful use of peroxidases in bleaching could are not yet convincingly managed because of the problem of peroxi dase inactivation by hydrogen peroxide or the appropriate Dosing technology for hydrogen peroxide in a non-continuous Technical processes have not been possible so far.

During the sole use of laccase the lignin does not enter the desired extractable form was transferred by adding ge suitable mediator substances for laccase a technical process be developed which pulp of any kind successfully can delignify. Such an enzymatic delignification stem, which consists of the components laccase, mediator and oxygen exists, is described in WO 94/29510. This registration gives also detailed the state of the art again.

Additional mediators for enzymatic delignifications at The use of laccase is described in WO 97/06244.

WO 94/01538 describes the use of cellobiose oxidase in Combination with an endoglycanase, for example xylanase and / or oxidoreductase, for example laccase. In the process a bleach booster is also added. The on given conditions call, among other things, an alkaline PH value. The effectiveness of such a system is very low because at the pH mentioned, the laccase described largely in is active. Laccase is also present in the presence of hydrogen peroxide deactivated. Added to this is the fact that they are called bleach boosters Connections show no amplifier activity. This is over WO 96/12846 shows where one of the cellulose bleaches analogous to An application, the bleaching of dyed textile fibers becomes:  

In Example 1, WO 96/12846 describes the difference in effectiveness mediators. As shown in Table 2 in this example Lich, the exemplary connections mentioned there show none Mediator activity.

WO 94/29510 describes the advantageous combination of an enzyme table delignification system consisting of a laccase and egg an active mediator. This method is currently the only one GE known enzymatic process, which leads to an effective Degradation of lignin resulted. N-OH-benzotriazole is used as a mediator (HBT) used. In the described process, among others rem also the addition of further enzymatic components, such as. B. Proteases mentioned.

As suggested by Oksanen et al. Shown in 1997 shows a combined instruction Use of xylanase / laccase / HBT only a very small improvement compared to a xylana-free application. Benefits erga only practice after the xylanase treatment from the lacca se / HBT treatment was separated and as a separate procedure step was carried out.

It is therefore desirable to use a multi-component system change, breakdown or bleaching of lignin and lignin-containing materials lien to have available, which better results in the Delignification is achieved as a known enzymatic delignification systems and not with the disadvantages of chemical deligni systems is affected.

In addition to the poor performance of the individual enzymatic ver drive to pulp delignification which is due to the above described problems necessary separation of enzymatic Delignification levels and the resulting gradual Procedure for the delignification of pulp serious disadvantage of these procedures  Need for washing and extraction steps between the The processes can be different enzyme treatments not economically in the existing delignification and Integrate bleaching sequences.

It is therefore also desirable to use enzymatic methods Treatment of lignin and lignin-containing materials ready for represent several enzymatic process steps in one unite single process stage.

The present invention relates to a multi-component system for Changing, breaking down or bleaching lignin, lignin-containing mate rialien or similar substances containing an oxidoreductase, an oxidizing agent suitable for the oxidoreductase, a Me diator and at least one enzymatically active additive, thereby characterized in that the mediator the oxidoreductase and the en cymatically active additive is not inactivated and that enzymatically effective additive from the group of hydrolases of the enzyme class 3.2.1. is selected.

The enzyme classes are named in the present application according to the International Enzyme Nomenclature, Committee of the International Union of Biochemistry and Molecular Biology (Enzyme Nomenclature, Academic Press, Inc., 1992, pp. 24-154).

In the sense of the invention, a mediator inactivates an enzyme if he lost <70% of the enzyme activity of this enzyme in within an incubation period of 30 min. in a test system which acts in a total volume of 50 ml at 45 ° C 60 IU Oxidoreductase, for example laccase and 400 IU hydrolase Enzyme class 3.2.1, for example xylanase, cellulase or a corresponding amount of another enzyme, as well as 7.5 mmol / l media gate contains.  

The multi-component system according to the invention has the following advantages over the prior art:

• - It has a high selectivity for lignin
• - It does not adversely affect fiber quality
• - It enables in combination with chemical delignification process a chemical saving in the overall delignification and bleaching
• - It represents an improvement of the previous individual procedures of Xylanase treatment and laccase mediator system. The Ver improvement is greater than the sum of the individual processes.

The use of cellulase in cell manufacturing processes So far, substance is unknown. Because cellulases are known to be cellulosic break them down and, according to previous opinion, always pulp damage, is their use in the process for the production of cells fabric completely unexpected and surprising for the expert.

Multicomponents can be used as oxidoreductases in the invention system Oxidoreductases of classes 1.1.1 to 1.97 according to internals tional enzyme nomenclature, Committee of the International Union of Biochemistry and Molecular Biology (Enzyme Nomenclature, Aca demic Press, Inc., 1992, pp. 24-154) can be used.

Oxidoreductases are preferably those mentioned below Classes used:

Class 1.1 enzymes, which comprise all dehydrogenases which act on primary, secondary alcohols and semiacetals, and which are accepted as NAD⁺ or NADP⁺ (subclass 1.1.1), cytochrome (1.1.2), oxygen (O ₂ ) ( 1.1.3), disulfides (1.1.4), quinones (1.1.5) or other acceptors (1.1.99).

The enzymes of the class are particularly preferred from this class 1.1.5 with quinones as acceptors and the enzymes of class 1.1.3 with oxygen as the acceptor.

Cellobiose is particularly preferred in this class: quinone-1-oxidoreductase (1.1.5.1).

Enzymes of class 1.2 are also preferred. This class of enzymes includes such enzymes that aldehydes to the corresponding acid or oxidize oxo groups. The acceptors can be NAD⁺, NADP⁺ (1.2.1), cytochrome (1.2.2), oxygen (1.2.3), sulfides (1.2.4), Iron-sulfur proteins (1.2.5) or other acceptors (1.2.99) be.

The enzymes of group (1.2.3) are particularly preferred here Oxygen as an acceptor.

Enzymes of class 1.3 are also preferred.

In this class, enzymes are summarized that are based on CH-CH group of the donor.

The corresponding acceptors are NAD⁺, NADP⁺ (1.3.1), Cytochro me (1.3.2), oxygen (1.3.3), quinones or related compounds genes (1.3.5), iron-sulfur proteins (1.3.7) or other accepto ren (1.3.99).

Bilirubin oxidase (1.3.3.5) is particularly preferred.

Here are also the enzymes of class (1.3.3) with oxygen as acceptor and (1.3.5) with quinones etc. as acceptor in particular prefers.  

Also preferred are class 1.4 enzymes which act on CH-NH ₂ groups of the donor.

The corresponding acceptors are NAD⁺, NADP⁺ (1.4.1), Cytochro me (1.4.2), oxygen (1.4.3), disulfide (1.4.4), iron-weld fel proteins (1.4.7) or other acceptors (1.4.99).

Enzymes of class 1.4.3 are also particularly preferred here Oxygen as an acceptor.

Also preferred are class 1.5 enzymes which are based on CH-NH group of the donor. The corresponding acceptors are NAD⁺, NADP⁺ (1.5.1), oxygen (1.5.3), disulfides (1.5.4), Quinones (1.5.5) or other acceptors (1.5.99).

Here too, enzymes with oxygen (O ₂ ) (1.5.3) and with chinos (1.5.5) are particularly preferred as acceptors.

Enzymes of class 1.6 which are based on NADH or NADPH work.

The acceptors here are NADP⁺ (1.6.1), heme proteins (1.6.2), disulfide (1.6.4), quinones (1.6.5), NO ₂ groups (1.6.6) and a flavin (1.6.8 ) or some other acceptors (1.6.99).

Enzymes of class 1.6.5 with chino are particularly preferred here as acceptors.

Also preferred are class 1.7 enzymes which act as donors on other NO ₂ compounds and as acceptors cyto chrome (1.7.2), oxygen (O ₂ ) (1.7.3), iron-sulfur proteins (1.7.7) or others (1.7.99).

Class 1.7.3 with oxygen as is particularly preferred here Acceptor.

Also preferred are class 1.8 enzymes which act as donors on sulfur groups and acceptors NAD⁺, NADP⁺ (1.8.1), cytochromes (1.8.2), oxygen (O ₂ ) (1.8.3), disulfides (1.8 .4), quinones (1.8.5), iron-sulfur proteins (1.8.7) or others (1.8.99).

Class 1.8.3 with oxygen (O ₂ ) and (1.8.5) with quinones as acceptors is particularly preferred.

Also preferred are class 1.9 enzymes, which act on donor groups and have oxygen (O ₂ ) (1.9.3), NO ₂ compounds (1.9.6) and others (1.9.99) as acceptors.

Group 1.9.3 with oxygen (O ₂ ) as acceptor (cytochrome oxidases) is particularly preferred here.

Also preferred are class 1.12 enzymes based on water act as a donor.

The acceptors are NAD⁺ or NADP⁺ (1.12.1) or others (1.12.99).

Enzymes of class 1.13 and 1.14 (oxi recover).

Furthermore, preferred enzymes are those of class 1.15 which are based on Superoxide radicals act as acceptors.

Superoxide dismutase (1.15.1.1) is particularly preferred here.

Enzymes of class 1.16 are also preferred.  

NAD⁺ or NADP⁺ (1.16.1) or oxygen (O ₂ ) (1.16.3) act as acceptors.

Enzymes of class 1.16.3.1 are particularly preferred here (Ferroxidase, e.g. ceruloplasmin).

Further preferred enzymes are those of group 1.17 (action on CH ₂ groups which are oxidized to -CHOH-), 1.18 (action on reduced ferredoxin as donor), 1.19 (action on reduced flavodoxin as donor) and 1.97 (others Oxidoredukta sen) belong.

The enzymes of group 1.11 are also particularly preferred. which act on a peroxide as an acceptor. This only subclass (1.11.1) contains the peroxidases.

The cytochrome C peroxidases are particularly preferred here (1.11.1.5), Catalase (1.11.1.6), the peroxidase (1.11.1.6), the Iodide peroxidase (1.11.1.8), the glutathione peroxidase (1.11.1.9), the chloride peroxidase (1.11.1.10), the L-ascorbate Peroxidase (1.11.1.11), the phospholipid hydroperoxide glutathio ne-peroxidase (1.11.1.12), the manganese peroxidase (1.11.1.13), the Diarylpropane peroxidase (ligninase, lignin peroxidase) (1.11.1.14).

Enzymes of class 1.10 which are based on Bi phenols and related compounds work. They catalyze that Oxidation of biphenols and ascorbates. Act as acceptors NAD⁺, NADP⁺ (1.10.1), cytochrome (1.10.2), oxygen (1.10.3) or others (1.10.99).

Of these in turn, class 1.10.3 enzymes with oxygen (O ₂ ) as the acceptor are particularly preferred.

Of the enzymes in this class, the enzymes are catechol oxidase (Tyrosinase) (1.10.3.1), L-ascorbate oxidase (1.10.3.3), o-amino phenol oxidase (1.10.3.4) and laccase (benzene diol: oxigen oxido reductase) (1.10.3.2) is preferred, the laccases (benzene diol: Oxigen oxidoreductase) (1.10.3.2) are particularly preferred.

The oxidoreductases mentioned are commercially available or read win according to standard procedures. As organisms to the pro plants, animal cells, for example oils, bacteria and fungi. Basically, both naturally occurring as well as genetically modified organisms Be an enzyme producer. Parts of unicellular or multicellular organisms conceivable as enzyme producers, especially Cell cultures.

For the particularly preferred oxidoreductases, such as those from the Group 1.11.1, but above all 1.10.3, and especially for the Pro Production of laccases are, for example, white rot fungi, such as Pleurotus, Phlebia and Trametes.

The multi-component system according to the invention comprises at least one oxidizing agent. Examples of oxidizing agents are air, oxygen, ozone, H ₂ O ₂ , organic peroxides, peracids such as peracetic acid, performic acid, persulfuric acid, persalpic acid, metachloroperoxibenzoic acid, perchloric acid, perborates, peracetates, persulfates, peroxides or oxygen species and their radicals such as OH, OOH , Singlet oxygen, superoxide (O ₂ ⁻), ozo nid, dioxygenyl cation (O ₂ ⁺), dioxiranes, dioxetanes or Fremy Radicals are used.

Oxidizing agents which ent are preferably used are not generated by the corresponding oxidoreductases can, e.g. B. dioxiranes from laccases plus carbonyls, or the  chemically regenerate the mediator or implement it directly can.

For example, peroxidases require hydrogen as the cosubstrate peroxide. This connection can either be directly into the system be given or released from a predecessor connection the. Finally, hydrogen peroxide can also be generated in situ by a Auxiliary reaction are formed, e.g. B. by the enzymatic order set of glucose with glucose oxidase (E.C. 1.1.3.4).

For example, oxidases use Sauer as the electron acceptor material. Oxygen is usually present in sufficient quantities dissolved aqueous solutions. However, oxygen can also be used nete measures such as stirring, use of oxygen gas or that Apply pressure to be introduced into the reaction solution. This is particularly necessary if the procedure is elevated Temperature should be operated because the solubility of acid substance in aqueous solutions decreases with increasing temperature.

The multi-component system according to the invention comprises at least one NEN mediator, the oxidoreductase and the enzymatically effective Additive not deactivated.

The mediator is preferably at least one compound from the Group of aliphatic, cycloaliphatic, heterocyclic or aromatic compounds selected at least one Contains N-hydroxy, oxime, nitroso, N-oxyl or N-oxi function, being substituted or unsubstituted 1-hydroxy-1-benzotriazole, 3H-benzotriazole-1-oxide and 2H-Benzotriazol-1-oxide are excluded.

Examples of such compounds are those mentioned below Compounds of formula I, II, or III, wherein the compounds of formulas II and III are preferred.  

Compounds of the general formula I are:

where X represents one of the following groups:
(-N = CR ⁴ -) _p , (-CR ⁴ = N-) _p , (-CR ⁵ = CR ⁶ ) _p
and p is 1 or 2,
wherein the radicals R ¹ to R ^{6 may be the} same or different and may independently represent one of the following groups: hydrogen, halogen, hydroxy, formyl, carboxy and salts and esters thereof, amino, nitro, C ₁ -C ₁₂ alkyl , C ₁ -C ₆ alkyloxy, carbonyl-C ₁ -C ₆ alkyl, phenyl, sulfono, esters and salts thereof, sulfamoyl, carbamoyl, phospho, phosphono, phosphonooxy and their salts and esters, the amino, carbamoyl and sulfamoyl groups of the radicals R ¹ to R ^{6 can} furthermore be unsubstituted or mono- or disubstituted by hydroxy, C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy,
and wherein the radicals R ² and R ³ can form a common group -A- and -A- represents one of the following groups:
(-CR ⁷ -CR ⁸ -CR ⁹ = CR ¹⁰ -) or (-CR ¹⁰ = CR ⁹ -CR ⁸ = CR ⁷ -).

The radicals R ⁷ to R ¹⁰ can be identical or different and, independently of one another, represent one of the following groups: hydrogen, halogen, hydroxy, formyl, carboxy and salts and esters thereof, amino, nitro, C ₁ -C ₁₂ alkyl, C ₁ -C ₆ -alkyloxy, carbonyl-C ₁ -C ₆ -alkyl, phenyl, sulfono, esters and salts thereof, sulfamoyl, carbamoyl, phospho, phosphono, phosphonooxy and their salts and esters, the amino, carbamoyl and Sulfamoyl groups of the radicals R ⁷ to R ^{10 may} furthermore be unsubstituted or mono- or disubstituted by hydroxy, C ₁ -C ₃ -alkyl, C ₁ -C ₃ -alkoxy and where the C ₁ -C ₁₂ -alkyl- , C ₁ -C ₆ alkyloxy, carbonyl-C ₁ -C ₆ alkyl, phenyl, aryl groups of the radicals R ⁷ to R ^{10 are} unsubstituted or can furthermore be substituted one or more times with the radical R ¹¹ and where the radical R ^{11 can be} one of the following groups: hydrogen, halogen, hydroxyl, formyl, carboxy and their salts and esters, amino, nitro, C ₁ -C ₁₂ alkyl, C ₁ -C ₆ -alkyloxy, carbonyl-C ₁ -C ₆ -alkyl, phenyl, aryl, and their esters and salts, the carbamoyl, sulfamoyl, amino groups of the radical R ^{11 being} unsubstituted or furthermore mono- or disubstituted with the radical R ¹² may be substituted and wherein R ¹² may represent one of the following groups: hydrogen, hydroxyl, formyl, carboxyl and salts and esters thereof, amino, ni tro, C ₁ -C ₁₂ alkyl, C ₁ -C ₆ - alkyloxy, carbonyl-C ₁ -C ₆ alkyl, phenyl, aryl.

Compounds of the general formula II are:

where X represents one of the following groups:
(-N = CR ⁴ -) _p , (-CR ⁴ = N-) _p , (-CR ⁵ = CR ⁶ ) _p
and p is 1 or 2.

The radicals R ¹ and R ⁴ to R ¹⁰ can be the same or different and, independently of one another, represent one of the following groups:
Hydrogen, halogen, hydroxy, formyl, carboxy and salts and esters thereof, amino, nitro, C ₁ -C ₁₂ alkyl, C ₁ -C ₆ alkyloxy, carbonyl-C ₁ -C ₆ alkyl, phenyl, aryl, sulfono , Esters and salts there of, sulfamoyl, carbamoyl, phospho, phosphono, phosphonooxy and their salts and esters, wherein the amino, carbamoyl and sulfamoyl groups of the radicals R ¹ and R ⁴ to R ¹⁰ continue to be unsubstituted or mono- or can be substituted twice with hydroxy, C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy
and wherein the C ₁ -C ₁₂ alkyl, C ₁ -C ₆ alkyloxy, carbonyl C ₁ -C ₆ alkyl, phenyl, aryl, aryl C ₁ -C ₆ alkyl groups of can be R ¹ and R ⁴ is unsubstituted by R ¹⁰ or additionally monosubstituted or polysubstituted by the radical R ¹² and wherein R ¹² may represent one of the following groups: hydrogen, halogen, hydroxy, formyl, carboxy and their salts and esters, amino, nitro, C ₁ -C ₁₂ alkyl, C ₁ -C ₆ alkyloxy, carbonyl-C ₁ -C ₆ alkyl, phenyl, aryl, sulfono, sulfeno, sulfino and their esters and salts
and wherein the carbamoyl, sulfamoyl, amino groups of the radical R ^{12 can be} unsubstituted or can furthermore be mono- or disubstituted by the radical R ¹³ and the radical R ^{13 can be} one of the following groups: hydrogen, hydroxy, formyl , Carboxy and their salts and esters, amino, nitro, C ₁ -C ₁₂ alkyl, C ₁ -C ₆ alkyloxy, carbonyl-C ₁ -C ₆ alkyl, phenyl, aryl.

Examples of the compounds mentioned are:

1-hydroxy-benzimidazole

1-hydroxybenzimidazole-2-carboxylic acid
1-hydroxybenzimidazole
2-methyl-1-hydroxybenzimidazole
2-phenyl-1-hydroxybenzimidazole.

1-hydroxyindoles

2-phenyl-1-hydroxyindole.

Substances of the general formula III are:

where X represents one of the following groups:
(-N = CR ⁴ -) _m , (-CR ⁴ = N-) _m , (-CR ⁵ = CR ⁶ -) _m
and m is 1 or 2.

What has been said above applies to the radicals R ⁷ to R ¹⁰ and R ⁴ to R ⁶ .

R ¹⁴ can be: hydrogen, C ₁ -C ₁₀ alkyl, C ₁ -C ₁₀ alkylcarbonyl, their C ₁ -C ₁₀ alkyl and C ₁ -C ₁₀ alkylcarbonyl unsubstituted or with one or more R ¹⁵ radicals can be substituted, where R ^{15 can represent} one of the following groups: hydrogen, halogen, hydroxyl, formyl, carboxy and salts and esters thereof, amino, nitro, C ₁ -C ₁₂ alkyl, C ₁ - C ₆ -alkyloxy, carbonyl-C ₁ -C ₆ -alkyl-, phenyl-, sulfono-, their esters and salts, sulfamoyl-, carbamoyl-, phospho-, phosphono-, phosphonooxy- and their salts and esters, where the amino -, carbamoyl and sulfamoyl groups of the radical R ^{15 may} furthermore be unsubstituted or mono- or disubstituted by hydroxy-, C ₁ -C ₃ -alkyl-, C ₁ -C ₃ -alkoxy-.

The mediator can preferably also be selected from the group of cyclic N-hydroxy compounds with at least one optionally substituted five or six-membered ring containing the structure mentioned in formula IV

and their salts, ethers or esters, where
B and D are the same or different and are O, S, or NR ¹⁶ , where
R ^{16 is} hydrogen, hydroxyl, formyl, carbamoyl, sulfono, ester or salt of sulfono, sulfamoyl, nitro, amino, phenyl, aryl-C ₁ -C ₅ alkyl, C ₁ -C ₁₂ -Alkyl-, C ₁ -C ₅ -alkoxy-, C ₁ -C ₁₀ -carbonyl-, carbonyl-C ₁ -C ₆ -alkyl-, phospho-, phosphono-, phosphonooxy radical, ester or salt of the phosphonooxy radical, where carbamoyl -, Sulfamoyl, amino and phenyl radicals unsubstituted or substituted one or more times with a radical R ¹⁷ and the aryl-C ₁ -C ₅ alkyl, C ₁ -C ₁₂ alkyl, C ₁ -C ₅ -alkoxy-, C ₁ -C ₁₀ -carbonyl, carbonyl-C ₁ -C ₆ -alkyl radicals may be saturated or unsaturated, branched or unbranched and may be substituted one or more times with a radical R ¹⁷ , where R ^{17 is the} same or different and is hydroxyl, formyl, carboxy, ester or salt of carboxy, carbamoyl, sulfono ester or salt of sulfono, sulfamoyl, nitro, amino, phenyl, C ₁ -C ₅ - Alkyl, C ₁ -C ₅ alkoxy means.

The mediator is preferably selected from the group of compounds of the general formula V, VI, VII or VIII,

where B, D have the meanings already mentioned and
the radicals R ¹⁸ -R ^{33 are} identical or different and have a halogen radical, carboxy radical, salt or ester of a carboxy radical or have the meanings mentioned for R ¹⁶ ,
wherein R ²⁴ and R ²⁵ or R ²⁶ and R ^{27 may} not simultaneously mean hydroxyl or amino radical and
optionally two of the substituents R ¹⁸ -R ²¹ , R ²² -R ²³ , R ²⁴ -R ²⁷ , R ²⁸ -R ³³ can be linked to form a ring -E-, where -E- has one of the following meanings:
(-CH = CH) - _n with n = 1 to 3, -CH = CH-CH = N- or

and where appropriate the radicals R ²⁴ -R ^{27 can} also be connected to one another by one or two bridge elements -F-, where -F- is identical or different and has one of the following meanings: -O-, -S, -CH ₂ -, -CR ³⁴ = CR ³⁵ -;
where R ³⁴ and R ^{35 are the} same or different and have the meaning of R ¹⁸ .

Compounds of the general are particularly preferred as mediators my formulas V, VI, VII or VIII, in which B and D, O or S mean.

Examples of such compounds are N-hydroxy-phthalimide and optionally substituted N-hydroxy-phthalimide derivatives, N-hydroxymaleimid and optionally substituted N-hydroxymaleimide derivatives, N-hydroxy-naphthalimide and optionally substituted N-hydroxy-naphthalimide Derivatives, N-hydroxysuccinimide and optionally substituted N-hydroxysuccinimide derivatives, preferably those in which the radicals R ²⁴ -R ^{27 are} polycyclically linked.

Particularly preferred mediators are N-hydroxyphthalimide, 4-amino-N-hydroxyphthalimide and 3-amino-N-hydroxyphthalimide.

Compounds of the formula V which are suitable as mediators are, for example:
N-hydroxyphthalimide,
4-amino-N-hydroxyphthalimide,
3-amino-N-hydroxyphthalimide,
N-hydroxy-benzene-1,2,4-tricarboximide,
N, N'-dihydroxy-pyromellitic diimide,
N, N'-dihydroxy-benzophenone-3,3 ', 4,4'-tetracarboxylic acid diimide.

Compounds of the formula VI which are suitable as mediators are, for example:
N-hydroxymaleimide,
Pyridine-2,3-dicarboxylic acid N-hydroxyimide.

Compounds of the formula VII which are suitable as mediators are, for example:
N-hydroxysuccinimide,
N-hydroxy tartarimide,
N-hydroxy-5-norbornene-2,3-dicarboximide,
exo-N-hydroxy-7-oxabicyclo [2.2.1] hept-5-ene-2,3-dicarboximide,
N-hydroxy-cis-cyclohexane-1,2-dicarboximide,
N-hydroxy-cis-4-cyclohexene-1,2-dicarboximide.

A suitable compound of formula VIII as a mediator is, for example:
N-hydroxynaphthalimide sodium salt.

A suitable compound with a six-membered ring containing the structure mentioned in formula IV is, for example:
N-hydroxyglutarimide.

The compounds mentioned by way of example are also suitable in the form their salts or esters as mediators.

Compounds selected from are also suitable as mediators the group of N-aryl-N-hydroxy amides.

Of these, compounds of the general formula IX, X or XI are preferably used as mediators

and their salts, ethers or esters, where
G monovalent homo- or heteroaromatic one- or two-nucleus radical and
L means double-bonded homo- or heteroaromatic mono- or dinuclear radical and
these aromatics by one or more, identical or different radicals R ³⁶ , selected from the group halogen, hydroxyl, formyl, cyano, carbamoyl, carboxy, ester or salt of carboxy, sulfono, ester or salt of sulfono residues, sulfamoyl, nitro, nitroso, amino, phenyl, aryl-C ₁ -C ₅ alkyl, C ₁ -C ₁₂ alkyl, C ₁ -C ₅ alkoxy, C ₁ -C _10- carbonyl, carbonyl-C ₁ -C ₆ -alkyl, phospho-, phosphono-, phosphono-oxy, ester or salt of the phosphonooxy may be substituted and
where carbamoyl, sulfamoyl, amino and phenyl radicals can be unsubstituted or substituted one or more times with a radical R ³⁷ and the aryl-C ₁ -C ₅ -alkyl-, C ₁ -C ₁₂ -alkyl-, C ₁ -C ₅ alkoxy, C ₁ -C ₁₀ carbonyl, carbonyl C ₁ -C ₆ alkyl radicals may be saturated or unsaturated, branched or unbranched and may be substituted one or more times with a radical R ³⁷ , in which
R ^{37 is the} same or different and hydroxyl, formyl, cyano, carboxy, ester or salt of carboxy, carbamoyl, sulfo no, sulfamoyl, nitro, nitroso, amino, phenyl, C ₁ -C ₅ alkyl, C ₁ -C ₅ alkoxy, C ₁ -C ₅ alkyl carbonyl radical and
two residues R ³⁶ or R ^{37 can be} linked in pairs over a bridge [-CR ³⁸ R ³⁹ -) _m with m equal to 0, 1, 2, 3 or 4 and
R ³⁸ and R ^{39 are the} same or different and are carboxy, ester or salt of carboxy, phenyl, C ₁ -C ₅ alkyl, C ₁ -C ₅ alkoxy, C ₁ -C ₅ alkylcarbonyl and one or more non-adjacent groups [-CR ³⁸ R ³⁹ -] by oxygen, sulfur or an imino residue optionally substituted with C ₁ to C ₅ alkyl and two adjacent groups [-CR ³⁸ R ³⁹ -] by a group [-CR ³⁸ = CR ³⁹ -] can be replaced and
I present in amidic form, monovalent acid residue of acids, selected from the group carboxylic acid with up to 20 carbon atoms, carbonic acid, half ester of carbonic acid or carbamic acid, sulfonic acid, phosphonic acid, phosphoric acid, monoester of phosphoric acid, diester of phosphoric acid and
K present in amidic form, double-bonded acid residue of acids, selected from the group consisting of mono- and dicarboxylic acids with up to 20 carbon atoms, carbonic acid, sulfonic acid, phosphonic acid, phosphoric acid, monoesters of phosphoric acid.

Particularly preferred mediators are compounds of the general formula XII, XIII, XIV, XV or XVI:

and their salts, ethers or esters, where
Ar ¹ monovalent homo- or heteroaromatic mononuclear aryl residue and
Ar ^{2 means} double-bonded homo- or heteroaromatic mononuclear aryl radical,
which by one or more, identical or different radicals R ⁴² , selected from the group hydroxyl, cyano, carboxy, ester or salt of carboxy, sulfono, ester or salt of sulfono, nitro, nitroso, amino, C ₁ -C ₁₂ alkyl, C ₁ -C ₅ alkoxy, C ₁ -C ₁₀ carbonyl, carbonyl C ₁ -C ₆ alkyl radicals can be substituted,
where amino radicals can be unsubstituted or substituted one or more times with a radical R ⁴³ and the C ₁ -C ₁₂ alkyl, C ₁ -C ₅ alkoxy, C ₁ -C ₁₀ carbonyl, carbonyl C ₁ - C ₆ -alkyl radicals can be saturated or unsaturated, branched or unbranched and can be substituted one or more times with a radical R ⁴³ ,
where R ^{43 is the} same or different and hydroxyl, carboxy, ester or salt of the carboxy, sulfono, nitro, amino, C ₁ -C ₅ alkyl, C ₁ -C ₅ alkoxy, C ₁ -C ₅ -Alkylcarbonylrest means and
each two R ⁴² radicals can be linked in pairs via a bridge [-CR ³⁸ R ³⁹ -] _m with m equal to 0, 1, 2, 3 or 4 and
R ³⁸ and R ^{39 have} the meanings already mentioned and one or more non-adjacent groups [-CR ³⁸ R ³⁹ -] by oxygen, sulfur or an imino radical which may be substituted by a C ₁ to C ₅ alkyl radical and two adjacent groups [- CR ³⁸ R ³⁹ -] can be replaced by a group [-CR ³⁸ = CR ³⁹ -],
R ⁴⁰ identical or different monovalent radicals selected from the group consisting of hydrogen, phenyl, aryl-C ₁ -C ₅ alkyl, C ₁ -C ₁₂ alkyl, C ₁ -C ₅ alkoxy, C ₁ -C ₁₀ denotes carbonyl radical, where phenyl radicals can be unsubstituted or substituted one or more times with a radical R ⁴⁴ and the aryl-C ₁ -C ₅ -alkyl-, C ₁ -C ₁₂ -alkyl-, C ₁ -C ₅ - Alkoxy, C ₁ -C ₁₀ carbonyl radicals can be saturated or unsaturated, branched or unbranched and can be substituted one or more times with a radical R ⁴⁴ , where
R ^{44 is the} same or different and is hydroxy, formyl, cyano, carboxy, ester or salt of carboxy, carbamoyl, sulfo no, sulfamoyl, nitro, nitroso, amino, phenyl, C ₁ -C ₅ denotes alkyl, C ₁ -C ₅ alkoxy and
R ^{41 is} divalent radicals selected from the group of ortho-, meta-, para-phenylene, aryl-C ₁ -C ₅ -alkyl, C ₁ -C ₁₂ -alkylene, C ₁ -C ₅ -alkylenedioxy radical, where Phenylene radicals unsubstituted or substituted one or more times with a radical R ⁴⁴ and the aryl-C ₁ -C ₅ alkyl, C ₁ -C ₁₂ alkyl, C ₁ -C ₅ alkoxy radicals saturated or unsaturated , can be branched or unbranched and can be substituted one or more times with a radical R ⁴⁴ , where
p represents 0 or 1 and
q represents an integer from 1 to 3.

Ar ^{1 is} preferably phenyl and
Ar ² ortho-phenylene radical, where Ar ¹ by up to five and Ar ² by up to four identical or different radicals selected from the group C ₁ -C ₃ alkyl, C ₁ -C ₃ alkylcarbonyl, carboxy, ester or Salt of the carboxy radical, sulfono radical, ester or salt of the sulfono radical, hydroxyl, cyano, nitro, nitroso and amino radicals can be substituted, wherein amino radicals with two different radicals selected from the group consisting of hydroxy and C ₁ -C ₃ -Alkylcarbonyl can be substituted.

R ^{40 is} preferably a monovalent radical selected from the group consisting of hydrogen, phenyl, C ₁ -C ₁₂ alkyl, C ₁ -C ₅ alkoxy radical, the C ₁ -C ₁₂ alkyl radicals and C ₁ -C ₅ alkoxy radicals saturated or un-saturated, branched or unbranched.

R ⁴¹ preferably denotes divalent radicals selected from the group of ortho- or para-phenylene, C ₁ -C ₁₂ alkylene, C ₁ -C ₅ alkylenedioxy radical, the aryl-C ₁ -C ₅ alkyl-, C ₁ -C ₁₂ alkyl, C ₁ -C ₅ alkoxy radicals can be saturated or unsaturated, branched or unbranched and can be substituted one or more times with a radical R ⁴⁴ .

R ⁴⁴ preferably denotes carboxy radical, ester or salt of car boxyrest, carbamoyl, phenyl, C ₁ -C ₃ alkoxy radical.

Examples of compounds used as mediators are N-hydroxyacetanilide, N-hydroxypivaloylanilide, N-hydroxyacrylanilide, N-hydroxybenzoylanilide, N-hydroxymethylsul fonylanilide, N-hydroxy-N-phenyl-methylcarbamate, N-hydroxy-3-oxo-butyrylanilide, N-hydroxy-4-cyanoacetanilide, N-hydroxy-4-methoxyacetanilide, N-hydroxyphenacetin,  N-hydroxy-2,3-dimethylacetanilide, N-hydroxy-2-methylacetanilide, N-hydroxy-4-methylacetanilide, 1-hydroxy-3,4-dihydroquinolin- (1H) -2-one, N, N'-dihydroxy-N, N'-diacetyl-1,3-phenylenediamine, N, N'-dihydroxy succinic acid dianilide, N, N'-dihydroxy-maleic acid dianilide, N, N'-dihydroxy-oxalic acid dianilide, N, N'-dihydroxy-phosphoric acid di anilide, N-acetoxyacetanilide, N-hydroxymethyloxalylanilide, N-hydroxymaleic acid monoanilide.

Preferred mediators are N-hydroxyacetanilide, N-hydroxyformanilide, N-hydroxy-N-phenylmethyl carbamate, N-hydroxy-2-methylacetanilide, N-hydroxy-4-methylacetanilide, 1-hydroxy-3,4-dihydroquinolin- (1H) -2-one and N-acetoxyacetani lid.

The mediator can also be selected from the group of N-alkyl-N-hydroxy-A- be selected.

Preferred mediators are compounds of the general formulas (XVII) or (XVIII)

as well as their salts, ethers or esters, where
M is the same or different and means monovalent, linear or ver branched or cyclic or polycyclic, saturated or unsaturated alkyl radical having 1-24 C atoms and
wherein this alkyl radical by one or more radicals R ⁴⁵ , which are the same or different and are selected from the group consisting of hydroxyl, mercapto, formyl, carbamoyl, carboxy, ester or salt of carboxy, sulfone, ester or salt of sulfono residues, sulfamoyl, nitro, nitroso, amino, hydroxylamino, phenyl, C ₁ -C ₅ alkoxy, C ₁ -C ₁₀ carbonyl, phospho-, phosphono-, phosphonooxy, ester or salt of Phosphonooxy residues can be substituted and
where carbamoyl, sulfamoyl, amino, hydroxylamino, mercapto and phenyl radicals can be unsubstituted or substituted one or more times with a radical R ⁴⁶ and the C ₁ -C ₅ alkoxy, C ₁ -C ₁₀ carbonyl radicals can be saturated or unsaturated, branched or unbranched and can be substituted one or more times with a radical R ⁴⁶ , where
R ^{46 is the} same or different and is hydroxy, formyl, cyano, carboxy, ester or salt of carboxy, carbamoyl, sulfo no, sulfamoyl, nitro, nitroso, amino, phenyl, benzoyl, C ₁ Means -C ₅ alkyl, C ₁ -C ₅ alkoxy, C ₁ -C ₅ alkyl carbonyl and not α-methylene groups can be replaced by oxygen, sulfur or an optionally monosubstituted imino residue and
N present in amidic form monovalent acid residue of acids, selected from the group of aliphatic or mono- or two-ring aromatic or mono- or dinuclear heteroaromatic carboxylic acids with up to 20 carbon atoms, carbonic acid, half-ester of carbonic acid or carbamic acid, sulfonic acid, Phosphonsäu re, phosphoric acid, monoesters of phosphoric acid, diesters of phosphoric acid means and
T present in amidic form, double-bonded acid residue of acids, selected from the group of aliphatic, mono- or dinuclear aromatic or mononuclear or dinuclear heteroaromatic dicarboxylic acids with up to 20 carbon atoms, carbonic acid, sulfonic acid, phosphonic acid, phosphoric acid, monoesters of phosphoric acid tet and
where alkyl radicals of the aliphatic acids N and T present in amidic form can be linear or branched and / or cyclic and / or polycyclic saturated or unsaturated and contain zero to 24 carbon atoms and are unsubstituted or mono- or polysubstituted by the radical R ⁴⁵ and
Aryl and heteroaryl radicals of the aromatic or heteroaromatic acids N and T present in amidic form by one or more radicals R ⁴⁷ , which are identical or different and are selected from the group consisting of hydroxyl, mercapto, formyl, cyano, carbamoyl , Carboxy, ester or salt of carboxy, sulfono, ester or salt of sulfono, sulfamoyl, nitro, nitroso, amino, phenyl, aryl-C ₁ -C ₅ alkyl, C ₁ -C ₁₂ alkyl, C ₁ -C ₅ alkoxy, C ₁ -C ₁₀ carbonyl, phospho-, phosphono-, phosphonooxy radical, ester or salt of the phosphonooxy radical can be substituted and
where carbamoyl, sulfamoyl, amino, mercapto and phenyl radicals can be unsubstituted or substituted one or more times with the radical R ⁴⁶ and the aryl-C ₁ -C ₅ -alkyl-, C ₁ -C ₁₂ -alkyl-C ₁ -C ₅ alkoxy, C ₁ -C ₁₀ carbonyl radicals may be saturated or unsaturated, branched or unbranched and may be substituted one or more times with the radical R ⁴⁶ .

Compounds with the general formulas (XIX, XX, XXI or XXII) are particularly preferred as mediators:

and their salts, ethers or esters, where
Alk ^{1 is} identical or different and means monovalent, linear or branched or cyclic or polycyclic, saturated or unsaturated alkyl radical having 1-10 C atoms, this alkyl radical being selected from one or more R ⁴⁸ radicals which are identical or different the group hydroxyl, formyl, carbamoyl, carboxy, ester or salt of car boxyrest, sulfono, ester or salt of sulfonorest, sulfamoyl, nitro, nitroso, amino, hydroxylamino, phenyl, C ₁ -C ₅ alkoxy, C ₁ -C ₅ carbonyl radicals can be substituted and wherein carbamoyl, sulfamoyl, amino, hydroxylamino and phenyl radicals can be unsubstituted or substituted one or more times with a radical R ⁴⁹ and the C ₁ -C ₅ alkoxy, C ₁ -C ₁₀ carbonyl radicals can be saturated or unsaturated, branched or unbranched and can be substituted one or more times with a radical R ⁴⁹ , where
R ^{49 is the} same or different and is hydroxyl, formyl, cyano, carboxy, ester or salt of carboxy, carbamoyl, sulfo no, sulfamoyl, nitro, amino, phenyl, benzoyl, C ₁ -C ₅ -Alkyl-, C ₁ -C ₅ -alkoxy-, C ₁ -C ₅ -alkylcarbonyl radical means and non-α-methylene groups can be replaced by oxygen, sulfur or an optionally monosubstituted imino radical and
where R ^{50 are} identical or different monovalent radicals selected from the group consisting of hydrogen, phenyl, pyridyl, furyl, pyrrolyl, thienyl, aryl-C ₁ -C ₅ -alkyl, C ₁ -C ₁₂ -alkyl -, C ₁ -C ₁₀ alkoxy, C ₁ -C ₁₀ carbonyl radical,
where phenyl, pyridyl, furyl, pyrrolyl and thienyl radicals can be unsubstituted or substituted one or more times with a radical R ⁵¹ and the aryl-C ₁ -C ₅ -alkyl-, C ₁ -C ₁₂ -alkyl -, C ₁ -C ₅ alkoxy and C ₁ -C ₁₀ carbonyl radicals can be saturated or unsaturated, branched or unbranched and can be substituted one or more times with a radical R ⁵¹ and
R ^{51 is the} same or different and hydroxyl, formyl, carboxy radical, ester or salt of the carboxy radical, carbamoyl, sulfono, sulfamoyl, nitro, amino, phenyl, C ₁ -C ₅ alkyl, C ₁ -C ₅ alkoxy radical means and
R ⁵² double-bonded radicals, selected from the group phenylene, pyridylene, thienylene, furylene, pyrrolylene, aryl-C ₁ -C ₅ alkyl, C ₁ -C ₁₂ alkylene, C ₁ -C ₅ -Alkylenedioxy radical, where phenylene, pyridylene, thienylene, furylene, pyrrolylene can be unsubstituted or substituted one or more times with a radical R ⁵³ and the aryl-C ₁ -C ₅ -alkyl-, C ₁ -C ₁₂ alkyl, C ₁ -C ₅ alkoxy radicals may be saturated or unsaturated, branched or unbranched and may be substituted one or more times with a radical R ⁵¹ , where
p means 0 or 1.

Particularly preferred mediators are compounds of the general formula (XIX-XXII) in which
Alk ^{1 is the} same or different and means monovalent, linear or branched or cyclic, saturated or unsaturated alkyl radical with 1-10 C atoms,
wherein this alkyl radical by one or more radicals R ⁴⁸ , which are the same or different and are selected from the group hydroxyl, carbamoyl, carboxy, ester or salt of carboxy, sulfono, ester or salt of sulfono, sulfamoyl, amino no -, Phenyl, C ₁ -C ₅ alkoxy, C ₁ -C ₅ carbonyl radicals can be substituted and
where carbamoyl, sulfamoyl, amino and phenyl radicals can be unsubstituted or substituted one or more times with a radical R ⁴⁹ and the C ₁ -C ₅ alkoxy, C ₁ -C ₁₀ carbonyl radicals saturated or unsaturated, branched or can be unbranched and can be substituted one or more times with a radical R ⁴⁹ , where R ^{49 is} identical or different and is hydroxyl, carboxy, ester or salt of carboxy, carbamoyl, sulfono-, sulfamoyl, nitro, amino -, Phenyl, benzoyl, C ₁ -C ₅ alkyl, C ₁ -C ₅ alkoxy, C ₁ -C ₅ alkylcarbonyl radical and
where R ^{50 is the} same or different monovalent radicals, selected from the group consisting of hydrogen, phenyl, furyl, aryl-C ₁ -C ₅ -alkyl-, C ₁ -C ₁₂ -alkyl-, C ₁ -C ₁₀ -alkoxy- , C ₁ -C ₁₀ carbonyl radical,
where phenyl and furyl radicals may be unsubstituted or mono- or polysubstituted by an R ⁵¹ radical and the aryl-C ₁ -C ₅ -alkyl-, C ₁ -C ₁₂ -alkyl-, C ₁ -C ₅ -alkoxy- and C ₁ -C ₁₀ carbonyl radicals can be saturated or unsaturated, branched or unbranched and can be substituted one or more times with a radical R ⁵¹ , where
R ^{51 is} identical or different and means carboxy radical, ester or salt of the carboxy radical, carbamoyl, phenyl, C ₁ -C ₅ alkyl, C ₁ -C ₅ alkoxy radical and
R ^{52 is a} double-bonded radical selected from the group consisting of phenylene, Fury len, C ₁ -C ₁₂ alkylene, C ₁ -C ₅ alkylenedioxy radical, phenylene and furanylene being unsubstituted or one or more than one radical R ⁵¹ can be substituted and the aryl-C ₁ -C ₅ alkyl, C ₁ -C ₁₂ alkyl, C ₁ -C ₅ alkoxy radicals may be saturated or unsaturated, ver branched or unbranched and with a radical R ⁵¹ can be substituted one or more times, where
p means 0 or 1.

Examples of compounds that can be used as mediators are
N-hydroxy-N-methyl-benzoic acid amide, N-hydroxy-N-methyl-benzenesulfonic acid amide, N-hydroxy-N-methyl-p-toluenesulfonic acid amide, N-hydroxy-N-methyl-furan-2-carboxylic acid amide, N -Hydroxy-N-methylthiophene-2-carboxamide, N, N'-dihydroxy-N, N'-dimethyl-phthalic acid diamide, N, N'-dihydroxy-N, N'-dimethyl-isophthalic acid diamide, N, N'- Dihydroxy-N, N'-dimethyl-terephthalic acid diamide, N, N'-dihydroxy-N, N'-dimethyl-benzene-1,3-disulfonic acid diamide, N, N'-dihydroxy-N, N'-dimethyl-furan-3 , 4-dicarboxylic acid diamide, N-hydroxy-N-tert-butyl-benzoic acid amide, N-hydroxy-N-tert-butyl-benzenesulfonic acid amide, N-hydroxy-N-tert-butyl-p-toluenesulfonic acid amide, N-hydroxy N-tert-butyl-furan-2-carboxylic acid amide, N-hydroxy-N-tert-butyl-thiophene-2-carboxylic acid amide, N, N'-dihydroxy-N, N'-di-tert-butyl-phthalic acid diamide , N, N'-dihydroxy-N, N'-di-tert-butyl-isophthalic acid diamide, N, N'-dihydroxy-N, N'-di-tert-butyl-terephthalic acid diamide, N, N'-dihydroxy- N, N'-di-tert-butyl-benzene-1,3-disulfonic acid diamide, N, N'-dihydroxy-N, N ' di-tert-butyl-furan-3,4-dicarboxylic acid diamide, N-hydroxy-N-cyclohexyl-benzoic acid amide, N-hydroxy-N-cyclohexyl benzenesulfonic acid amide, N-hydroxy-N-cyclohexyl-p-toluenesulfonic acid amide, N-hydroxy-N-cyclohexyl-furan-2-carboxylic acid amide, N-hydroxy-N-cyclohexyl-thiophene-2-carboxylic acid amide, N, N'-dihydroxy-N, N'-dicyclohexyl-phthalic acid diamide, N, N'-dihydroxy -N, N'-dicyclohexyl-isophthalic acid diamide, N, N'-dihydroxy-N, N'-dicyclohexyl-terephthalic acid diamide, N, N'-dihydroxy-N, N'-dicyclohexyl-benzene-1,3-disulfonic acid diamide, N , N'-dihydroxy-N, N'-dicyclohexyl-furan-3,4-dicarboxylic acid di amide, N-hydroxy-N-isopropyl-benzoic acid amide, N-hydroxy-N-isopropyl-benzene-sulfonic acid amide, N-hydroxy -N-isopropyl-p-toluenesulfonamide, N-hydroxy-N-isopropyl-furan-2-carboxamide, N-hydroxy-N-isopropyl-thiophene-2-carboxylic acid amide, N, N'-dihydroxy-N, N '-diisopropyl-phthalic acid diamide, N, N'-dihydroxy-N, N'-diisopropyl-isophthalic acid diamide, N, N'-dihydroxy-N, N'-diisopropyl-terephthalic acid diamide, N, N'-dihy doxy-N, N'-diisopropyl-benzene-1,3-disulfonic acid diamide, N, N'-dihydroxy-N, N'-diisopropyl furan-3,4-dicarboxylic acid diamide, N-hydroxy-N-methyl-acetamide , N-Hydroxy-N-tert-butyl-acetamide, N-Hydroxy-N-isopropyl-acetamide, N-Hydroxy-N-cyclohexyl-acetamide, N-Hydroxy-N-methyl-pivalic acid amide, N-Hydroxy-N- isopropyl-pivalic acid amide, N-hydroxy-N-methyl-acrylamide, N-hydroxy-N-tert-butyl-acrylamide, N-hydroxy-N-isopropyl-acrylamide mid, N-hydroxy-N-cyclohexyl-acrylamide, N -Hydroxy-N-methyl-methane sulfonamide, N-hydroxy-N-isopropyl-methanesulfonamide, N-hydroxy-N-isopropyl-methylcarbamate, N-hydroxy-N-methyl-3-oxo-butyric acid amide, N, N'-dihydroxy -N, N'-di benzoyl-ethylenediamine, N, N'-dihydroxy-N, N'-dimethyl succinic acid diamide, N, N'-dihydroxy-N, N'-di-tert-butyl-maleic acid diamide, N-hydroxy -N-tert-butyl-maleic acid monoamide, N, N'-dihydroxy-N, N'-di-tert.-butyl-oxalic acid diamide, N, N'-dihydroxy-N, N'-di-tert.-butyl- phosphoric acid diamide.

Compounds are preferably selected as mediators from the Group N-hydroxy-N-methyl-benzoic acid amide, N-hydroxy-N-methyl benzenesulfonic acid amide, N-hydroxy-N-methyl-p-toluenesul formic acid amide, N-hydroxy-N-methyl-furan-2-carboxylic acid amide, N, N'-dihydroxy-N, N'-dimethyl-phthalic acid diamide, N, N'-dihydroxy-N, N'-di methyl terephthalic acid diamide, N, N'-dihydroxy-N, N'-dimethyl-benzene-1,3-disulfonic acid diamide, N-hydroxy-N-tert-butyl-benzoic acid amide, N-hydroxy-N-tert-butyl-benzenesulfonic acid amide, N-hydroxy-N-tert-butyl-p-toluenesulfonamide, N-hydroxy-N-tert-butyl-furan-2-carboxamide, N, N'-dihydroxy-N, N'-di-tert-butyl-terephthalic acid diamide, N-hydroxy-N-isopropyl-benzoic acid amide, N-hydroxy-N-isopropyl-p-to luolsulfonic acid amide, N-hydroxy-N-isopropyl-furan-2-carboxamide, N, N'-dihydroxy- N, N'-diisopropyl-terephthalic acid diamide, N, N'-dihydroxy-N, N'-dii sopropyl-benzene-1,3-disulfonic acid diamide, N-hydroxy-N-methyl-ace tamide, N-hydroxy-N-tert-butyl-acetamide, N-hydroxy-N-isopropyl acetamide, N-hydroxy-N-cyclohexyl-acetamide, N-hydroxy-N-methyl-pi valic acid amide, N-hydroxy-N-tert-butyl-acrylamide, N-hydroxy-N-i sopropyl acrylamide, N-hydroxy-N-methyl-3-oxo-butyric acid amide, N, N'-dihydroxy-N, N'-dibenzoyl-ethylenediamine, N, N'-dihydroxy-N, N'-di-tert-butyl-maleic acid diamide, N-hydroxy-N-tert-butyl-maleic acid monoamide, N, N'-dihydroxy-N, N'-di-tert-butyl-oxalic acid diamide.

The mediator can also be selected from the group of oximes of the general formula XXIII or XXIV

and their salts, ethers, or esters, where
U is the same or different and is O, S, or NR ⁵³ , where
R ^{53 is} hydrogen, hydroxyl, formyl, carbamoyl, sulfono, ester or salt of sulfono, sulfamoyl, nitro, amino, phenyl, aryl, C ₁ -C ₅ alkyl, C ₁ -C ₁₂ -Alkyl-, C ₁ -C ₅ -alkoxy-, C ₁ -C ₁₀ -carbonyl-, carbonyl-C ₁ -C ₆ -alkyl-, phospho-, phosphono-, phosphonooxy radical, ester or salt of the phosphonooxy radical,
where carbamoyl, sulfamoyl, amino and phenyl radicals can be unsubstituted or substituted one or more times with a radical R ⁵⁴ and the aryl-C ₁ -C ₅ -alkyl-, C ₁ -C ₁₂ -alkyl-, C ₁ -C ₅ alkoxy, C ₁ -C ₁₀ carbonyl, carbonyl C ₁ -C ₆ alkyl radicals may be saturated or unsaturated, branched or unbranched and may be substituted one or more times with a radical R ⁵⁴ , in which
R ^{54 is the} same or different and is hydroxyl, formyl, carboxy, ester or salt of carboxy, carbamoyl, sulfono ester or salt of sulfono, sulfamoyl, nitro, amino, phenyl, C ₁ -C ₅ -Alkyl-, C ₁ -C ₅ alkoxy radical means and
the radicals R ⁵⁵ and R ^{56 are} identical or different and are halogen, carboxy, ester or salt of the carboxy radical, or have the meanings given for R ⁵³ , or to form a ring [-CR ⁵⁹ R ⁶⁰ ] _n with n equal to 2, 3 or 4 are linked and
R ⁵⁷ and R ^{58 have} the meanings given for R ⁵³ and
R ⁵⁹ and R ^{60 are} identical or different and represent halogen, carboxy radical, ester or salt of the carboxy radical, or have the meanings mentioned for R ⁵³ .

Compounds with all are particularly preferred as mediators general formula XXIII in which U is O or S and the rest gene radicals have the meanings given above. A case The game for such a compound is 2-hydroxyiminomalonic acid dimethyl ester.

Isonitrosoderi are also particularly preferred as mediators vate of cyclic ureids of the general formula XXIV. Exispie le for such compounds are 1-methylvioluric acid, 1,3-dimethylvioluric acid, thiovioluric acid, alloxan-4,5-dioxime.

Alloxan-5-oxime hydrate is particularly preferred as mediator (Violuric acid) and / or its esters, ethers or salts.

The mediator can also be selected from the group of vicinal nitroso-substituted aromatic alcohols of the general formulas XXV or XXVI

and their salts, ethers or esters, where
R ⁶¹ , R ⁶² , R ⁶³ and R ^{64 are the} same or different and are hydrogen, halogen, hydroxy, formyl, carbamoyl, carboxy, ester or salt of carboxy, sulfono, ester or salt of sulfono, sulfamoyl- , Nitro, nitroso, cyano, amino, phenyl, arylC ₁ -C ₅ alkyl, C ₁ -C ₁₂ alkyl, C ₁ -C ₅ alkoxy, C ₁ -C ₁₀ - Carbonyl, carbonyl-C ₁ -C ₆ -alkyl, phospho-, phosphono-, phosphono-oxy radical, ester or salt of the phosphonooxy radical mean,
where carbamoyl, sulfamoyl, amino and phenyl radicals can be unsubstituted or substituted one or more times with a radical R ⁶⁵ and the aryl-C ₁ -C ₅ -alkyl-, C ₁ -C ₁₂ -alkyl-, C ₁ -C ₅ alkoxy, C ₁ -C ₁₀ carbonyl, carbonyl C ₁ -C ₆ alkyl radicals may be saturated or unsaturated, branched or unbranched and may be substituted one or more times with a radical R ⁶⁵ , in which
R ^{65 is the} same or different and is hydroxyl, formyl, carboxy, ester or salt of carbox, carbamoyl, sulfono, sul famoyl, nitro, nitroso, amino, phenyl, C ₁ -C ₅ - Alkyl, C ₁ -C ₅ alkoxy radical means or
the residues R ⁶¹ -R ⁶⁴ are linked in pairs to form a ring [-CR ⁶⁶ R ⁶⁷ -] _m , where m is an integer and has a value from 1 to 4, or to form a ring [-CR ⁶⁸ = CR ⁶⁹ -] _n are linked, where n is an integer and has a value from 1 to 3, and
R ⁶⁶ , R ⁶⁷ , R ⁶⁸ and R ^{69 are the} same or different and have the meanings given for R ⁶¹ to R ⁶⁴ .

Aromatic alcohols are preferably phenols or higher to understand condensed derivatives of phenol.  

Preferred mediators are compounds of the general formula XXV or XXVI, the synthesis of which is substituted for nitrosation ized phenols can be recycled. Examples of such connections genes are 2-nitrosophenol, 3-methyl-6-nitrosophenol, 2-methyl-6-nitrosophenol, 4-methyl-6-nitrosophenol, 3-ethyl-6-nitrosophenol, 2-ethyl-6-nitrosophenol, 4-ethyl-6-nitrosophenol, 4-isopropyl-6-nitrosophenol, 4-tert-butyl-6-nitrosophenol, 2-phenyl-6-nitrosophenol, 2-benzyl-6-nitrosophenol, 4-benzyl-6-nitrosophenol, 2-hydroxy-3-nitrosobenzyl alcohol, 2-hydroxy-3-nitrosobenzoic acid, 4-hydroxy-3-nitrosobenzoic acid, 2-methoxy-6-nitrosophenol, 3,4-dimethyl-6-nitrosophenol, 2,4-dimethyl-6-nitrosophenol, 3,5-dimethyl-6-nitrosophenol, 2,5-dimethyl-6-nitrosophenol, 2-nitrosoresorcin, 4-nitrosoresorcin, 2-nitrosoorcin, 2-nitrosophloroglucin and 4-nitrosopyrogallol, 4-nitroso-3-hydroxyaniline, 4-nitro-2-nitrosophenol.

Further preferred mediators are o-nitroso derivatives condensed aromatic alcohols. Examples of such a verb are 2-nitroso-1-naphthol, 1-methyl-3-nitroso-2-naphthol and 9-hydroxy-10-nitroso-phenanthrene.

Particularly preferred mediators are 1-nitroso-2-naphthol, 1-nitroso-2-naphthol-3,6-disulfonic acid, 2-nitroso-1-naphthol-4-sulfonic acid, 2,4-dinitroso-1,3-dihydroxybenzene and esters, ethers or salts of the connections mentioned.

The mediator can also be selected from the group Hydroxypyridines, aminopyridines, hydroxyquinolines, aminoquinolines, Hydroxyisoquinolines, aminoisoquinolines with the hydroxy or Amino groups in the ortho or para position of nitroso or mercapto substituents, tautomers of the compounds mentioned and their Salts, ethers and esters.  

Preferred mediators are compounds of the general formula (XXVII), (XXVIII) or (XXIX)

and tautomers, salts, ethers or esters of the abovementioned compounds are present, where in the formulas XXVII, XXVIII or XXIX two mutually ortho or para radicals R ^{70 are} hydroxyl and nitroso or hydroxyl and mercapto or nitroso and amino
and the other R ⁷⁰ radicals are the same or different and are selected from the group consisting of hydrogen, halogen, hydroxy, mercapto, formyl, cyano, carbamoyl, carboxy, ester and salt of carboxy, sulfono, ester and salt of the sulfono radical, sulfamoyl, nitro, nitroso, amino, phenyl, aryl-C ₁ -C ₅ alkyl, C ₁ -C ₁₂ alkyl, C ₁ -C ₅ alkoxy, C ₁ -C ₁₀ carbonyl, car bonyl-C ₁ -C ₆ alkyl, phospho-, phosphono-, phosphono-oxy, ester and salt of the phosphonooxy and
where carbamoyl, sulfamoyl, amino, mercapto and phenyl radicals can be unsubstituted or substituted one or more times with a radical R ⁷¹ and
the aryl-C ₁ -C ₅ alkyl, C ₁ -C ₁₂ alkyl, C ₁ -C ₅ alkoxy, C ₁ -C ₁₀ carbonyl, carbonyl-C ₁ -C ₆ alkyl radicals saturated or can be unsaturated, branched or unbranched and can be substituted one or more times with a radical R ⁷¹ , where R ^{71 is the} same or different and is hydroxyl, formyl, cyano, carboxy, ester or salt of carboxy, carbamoyl, Sulfono, sulfamoyl, nitro, nitroso, amino, phenyl, C ₁ -C ₅ alkyl, C ₁ -C ₅ alkoxy or C ₁ -C ₅ alkylcarbonyl radical means and two radicals R ⁷⁰ each or two residues R ⁷¹ or R ⁷⁰ and R ^{71 can be} linked in pairs over a bridge [-CR ⁷² R ⁷³ -] _m with m equal to 1, 2, 3 or 4 and
R ⁷² and R ^{73 are the} same or different and are carboxy, ester or salt of carboxy, - phenyl, C ₁ -C ₅ alkyl, C ₁ -C ₅ alkoxy or C ₁ -C ₅ alkylcarbonyl and one or several non-adjacent groups [-CR ⁷² R ⁷³ -] by oxygen, sulfur or an imino residue which may be substituted with C ₁ -C ₅ -alkyl and two adjacent groups [-CR ⁷² R ⁷³ -] by one group [-CR ⁷² = R ⁷³ -] can be replaced.

Compounds of the general formula (XXVII) or (XXVIII) and their tautomers, salts, ethers or esters are particularly preferred as mediators, and in the formulas (XXVII) and (XXVIII), two radicals R ⁷⁰ hydroxy which are ortho to one another are particularly preferred - and nitroso or hydroxy and mercapto or nitroso and amino mean and
the remaining R ⁷⁰ radicals are the same or different and are selected from the group consisting of hydrogen, hydroxyl, mercapto, formyl, carbamoyl, carboxy radical, ester and salt of carboxy radical, sulfo norest, ester and salt of sulfono radical, sulfamoyl, Nitro, nitroso, amino, phenyl, aryl C ₁ -C ₅ alkyl, C ₁ -C ₅ alkyl, C ₁ -C ₅ alkoxy, C ₁ -C ₅ carbonyl , Carbonyl-C ₁ -C ₆ alkyl, phospho-, phosphono-, phosphono-oxy radical, ester and salt of the phosphonooxy radical
Carbamoyl, sulfamoyl, amino, mercapto and phenyl radicals can be unsubstituted or substituted one or more times with a radical R ⁷¹ and
the aryl-C ₁ -C ₅ alkyl, C ₁ -C ₅ alkyl, C ₁ -C ₅ alkoxy, C ₁ -C ₅ carbonyl, carbonyl-C ₁ -C ₆ alkyl radicals can be saturated or unsaturated, branched or unbranched and can be substituted one or more times with a radical R ⁷¹ , where R ^{71 has} the meanings already mentioned and
two residues R ⁷¹ can be linked in pairs over a bridge [-CR ⁷² R ⁷³ -] _m with m equal to 2, 3 or 4 and
R ⁷² and R ^{73 have} the meanings already mentioned and one or more non-adjacent groups [-CR ⁷² R ⁷³ -] can be replaced by oxygen or an imino radical which is optionally substituted by C ₁ -C ₅ -alkyl.

Examples of compounds used as mediators can be 2,6-dihydroxy-3-nitrosopyridine, 2,3-dihydroxy-4-nitrosopyridine, 2,6-dihydroxy-3-nitrosopyridine-4-carboxylic acid, 2,4-dihydroxy-3-nitrosopyridine, 3-hydroxy-2-mercaptopyridine, 2-hydroxy-3-mercaptopyridine, 2,6-diamino-3-nitrosopyridine, 2,6-diamino-3-nitrosopyridine-4-carboxylic acid, 2-hydroxy-3-nitrosopyridine, 3-hydroxy-2-nitrosopyridine, 2-mercapto-3-nitrosopyridine, 3-mercapto-2-nitrosopyridine, 2-amino-3-nitrosopyridine, 3-amino-2-nitrosopyridine, 2,4-dihydroxy-3-nitrosoquinoline, 8-hydroxy-5-nitrosoquinoline, 2,3-dihydroxy-4-nitrosoquinoline, 3-hydroxy-4-nitrosoisoquinoline, 4-hydroxy-3-nitrosoisoquinoline, 8-hydroxy-5-nitrosoisoquinoline as well as tautomers of these compounds.

Preferred mediators are 2,6-dihydroxy-3-nitrosopyridine, 2,6-diamino-3-nitrosopyridine, 2,6-dihydroxy-3-nitrosopyridine-4-carboxylic acid, 2,4-dihydroxy-3-nitrosopyridine, 2-hydroxy-3-mercaptopyridine, 2-mercapto-3-pyridinol, 2,4-dihydroxy-3-nitrosoquinoline, 8-Hydroxy-5-nitrosoquinoline, 2,3-dihydroxy-4-nitrosoquinoline so like tautomers of these compounds.  

The mediator can also be selected from the group of stable nitroxyl radicals le (nitroxide) must be selected, d. H. these free radicals can preserved, characterized and stored in pure form.

Compounds of the general formula (XXX), (XXXI) or (XXXII) are preferably used as mediators

in which
Ar is a monovalent homo- or heteroaromatic one- or two-nucleus radical and
wherein this aromatic radical by one or more, identical or different radicals R ⁷⁵ , selected from the group halogen, formyl, cyano, carbamoyl, carboxy, ester or salt of car boxyrest, sulfonorest, ester or salt of sulfonorest, Sulfamoyl, nitro, nitroso, amino, phenyl, arylC ₁ -C ₅ alkyl, C ₁ -C ₁₂ alkyl, C ₁ -C ₅ alkoxy, C ₁ -C ₁₀ -Carbonyl-, carbonyl-C ₁ -C ₆ alkyl-, phospho-, phosphono-, phosphonooxy, ester or salt of Phos phonooxy can be substituted and
where phenyl, carbamoyl and sulfamoyl radicals can be unsubstituted or substituted one or more times with a radical R ⁷⁶ , the amino radical can be substituted once or twice with R ⁷⁶ and the aryl-C ₁ -C ₅ -alkyl, C ₁ - C ₁₂ alkyl, C ₁ -C ₅ alkoxy, C ₁ -C ₁₀ carbonyl, carbonyl C ₁ -C ₆ alkyl radicals may be saturated or unsaturated, branched or unbranched and may be with a radical R ⁷⁶ can be substituted one or more times, where
R ⁷⁶ may be present one or more times and is the same or different and hydroxyl, formyl, cyano, carboxy, ester or salt of the carboxy, carbamoyl, sulfono, sulfamoyl, nitro, nitroso, amino -, Phenyl, C ₁ -C ₅ alkyl, C ₁ -C ₅ alkoxy, C ₁ -C ₅ alkyl carbonyl group and
R ^{74 is the} same or different and is halogen, hydroxyl, mercapto, formyl, cyano, carbamoyl, carboxy, ester or salt of carboxy, sulfono, ester or salt of sulfono, sulfamoyl, nitro, nitroso , Amino, phenyl, aryl C ₁ -C ₅ alkyl, C ₁ -C ₁₂ alkyl, C ₁ -C ₅ alkoxy, C ₁ -C ₁₀ carbonyl, carbonyl C ₁ - C ₆ -alkyl, phospho-, phosphono-, phosphono-oxy radical, ester or salt of the phosphonooxy radical
and R ⁷⁴ in the case of bicyclic stable nitroxyl radicals (structure XXXII) can also mean hydrogen and
where carbamoyl, sulfamoyl, amino, mercapto and phenyl radicals can be unsubstituted or substituted one or more times with a radical R ⁷⁷ and the aryl-C ₁ -C ₅ -alkyl-, C ₁ -C ₁₂ -alkyl- , C ₁ -C ₅ alkoxy, C ₁ -C ₁₀ carbonyl, carbonyl-C ₁ -C ₆ alkyl radicals may be saturated or unsaturated, branched or unbranched and substituted one or more times with a radical R ⁷⁷ may be, wherein R ^{77 is the} same or different and hydroxyl, formyl, cyano, carboxy, ester or salt of carboxy, carbamoyl, sulfo no, sulfamoyl, nitro, nitroso, amino, phenyl, C ₁ -C ₅ alkyl, C ₁ -C ₅ alkoxy, C ₁ -C ₅ alkylcarbonyl and
two residues R ⁷⁶ or R ^{77 can be} linked in pairs over a bridge [-CR ⁷⁸ R ⁷⁹ -] _m with m equal to 0, 1, 2, 3 or 4 and
R ⁷⁸ and R ^{79 are the} same or different and halogen, carboxy, ester or salt of carboxy, carbamoyl, sulfamoyl, phenyl, benzoyl, C ₁ -C ₅ alkyl, C ₁ -C ₅ alkoxy, C ₁ -C ₅ alkylcarbonyl and be
one or more non-adjacent groups [-CR ⁷⁸ R ⁷⁹ -] by oxygen, sulfur or an imino residue optionally substituted with C ₁ -C ₅ alkyl- and two adjacent groups [-CR ⁷⁸ R ⁷⁹ -] by one group [ -CR ⁷⁸ = CR ⁷⁹ -], [-CR ⁷⁸ = N-] or [-CR ⁷⁸ = N (O) -] can be replaced.

Particularly preferred mediators are nitroxyl radicals of the general formulas (XXXIII) and (XXXIV)

in which
R ^{80 is} identical or different and is phenyl-, aryl-C ₁ -C ₅ -alkyl-, C ₁ -C ₁₂ -alkyl-, C ₁ -C ₅ -alkoxy-, C ₁ -C ₁₀ -carbonyl-, carbonyl- C ₁ -C ₆ alkyl
where phenyl radicals can be unsubstituted or substituted one or more times with a radical R ⁸² and the aryl-C ₁ -C ₅ -alkyl-, C ₁ -C ₁₂ -alkyl-, C ₁ -C ₅ -alkoxy-, C ₁ -C ₁₀ carbonyl, carbonyl C ₁ -C ₆ alkyl radicals may be saturated or unsaturated, branched or unbranched and may be substituted one or more times with a radical R ⁸² ,
where R ⁸² can be present one or more times and is the same or different and hydroxyl, formyl, carboxy, ester or salt of carboxy, carbamoyl, sulfono, sulfamoyl, nitro, nitroso, amino, phenyl, Benzoyl, C ₁ -C ₅ alkyl, C ₁ -C ₅ alkoxy, C ₁ -C ₅ alkylcarbonyl and
R ^{81 is} identical or different and is hydrogen, hydroxyl, mer capto, formyl, cyano, carbamoyl, carboxy, ester or salt of carboxy, sulfono, ester or salt of sulfono, sulfamoyl, nitro, nitroso , Amino, phenyl, aryl C ₁ -C ₅ alkyl, C ₁ -C ₁₂ alkyl, C ₁ -C ₅ alkoxy, C ₁ -C ₁₀ carbonyl, carbonyl C ₁ - C ₆ -alkyl, phospho-, phosphono-, phosphonooxy, ester or salt of Phos phono-oxyrest
where carbamoyl, sulfamoyl, amino, mercapto and phenyl radicals can be unsubstituted or substituted one or more times with a radical R ⁷⁶ and the aryl-C ₁ -C ₅ -alkyl-, C ₁ -C ₁₂ -alkyl- , C ₁ -C ₅ alkoxy, C ₁ -C ₁₀ carbonyl, carbonyl-C ₁ -C ₆ alkyl radicals may be saturated or unsaturated, branched or unbranched and substituted one or more times with a radical R ⁷⁶ can be and a [-CR ⁸¹ R ⁸¹ -] - group by oxygen, an optionally substituted with C ₁ -C ₅ alkyl imino residue, a (hydroxy) imino residue, a car bonylfunction or one optionally with R ⁷⁶ mono- or disubstituted vinylidene function can be replaced and
two adjacent groups [-CR ⁸¹ R ⁸¹ -] can be replaced by a group [-CR ⁸¹ = CR ⁸¹ -] or [-CR ⁸¹ = N-] or [-CR ⁸¹ = N (O) -].

Examples of compounds that can be used as mediators are
2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO), 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl, 4-oxo-2,2,6,6 -tetramethyl-piperidine-1-oxyl, 4-acetamido-2,2,6,6-tetramethyl-piperidine-1-oxyl, 4- (ethoxyfluorophosphinyloxy) -2,2,6,6-tetramethyl-piperidine-1-oxyl 4- (isothiocyanato) -2,2,6,6-tetramethyl-piperidine-1-oxyl, 4-maleimido-2,2,6,6-tetramethyl-piperidine-1-oxyl, 4- (4-nitrobenzoyloxy) - 2,2,6,6-tetramethyl-piperidine-1-oxyl, 4- (phosphonooxy) -2,2,6,6-tetramethyl-piperidine-1-oxyl, 4-cyano-2,2,6,6- tetramethyl-piperidine-1-oxyl, 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl, 4-phenyl-2,2,5,5-tetramethyl-3-imidazoline-3- oxide-1-oxyl, 4-carbamoyl-2,2,5,5-tetramethyl-3-imidazoline-3-oxide-1-oxyl, 4-phenacylidene-2,2,5,5-tetramethyl-imidazolidine-1- oxyl, 3- (aminomethyl) -2,2,5,5-tetramethyl-pyrrolidine-N-oxyl, 3-carbamoyl-2,2,5,5-tetramethyl-pyrrolidine-N-oxyl, 3-carboxy-2, 2,5,5-tetramethyl-pyrrolidine-N-oxyl, 3-cyano-2,2,5,5,5-tetramethyl-pyrrolidine-N-oxyl, 3-maleimido-2,2,5,5-tetramethyl-pyrrolidine N-oxyl, 3 - (4-nitrophenoxycarbonyl) -2,2,5,5-tetramethyl-pyrrolidine-N-oxyl.

Preferred mediators are preferred
2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO), 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl, 4-oxo-2,2,6,6 -tetramethyl-piperidine-1-oxyl, 4-acetamido-2,2,6,6-tetramethyl-piperidine-1-oxyl, 4- (isothiocyanato) -2,2,6,6-tetramethyl-piperidine-1-oxyl , 4-maleimido-2,2,6,6-tetramethyl-piperidine-1-oxyl, 4- (4-nitrobenzoyloxy) -2,2,6,6-tetramethyl-piperidine-1-oxyl, 4- (phosphonooxy) -2,2,6,6-tetramethyl-piperidine-1-oxyl, 4-cyano-2,2,6,6-tetramethyl-piperidine-1-oxyl, 3-carbamoyl-2,2,5,5-tetramethyl -3-pyrroline-1-oxyl, 4-phenyl-2,2,5,5-tetramethyl-3-imidazoline-3-oxide-1-oxyl, 4-carbamoyl-2,2,5,5-tetramethyl-3 -imidazoline-3-oxide-1-oxyl, 4-phenacylidene-2,2,5,5-tetramethyl-imidazolidin-1-oxyl.

Are particularly preferred as mediators 2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO), and 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl.

Avoiding harmful mediator influence from the Ge overall system can also be achieved in that a hydrolase the enzyme class 3.2.1 is used, which compared to a Mo activation by activated mediator none for the described Use adverse change in their enzymatic properties shows (e.g., a mediator resistant xylanase or cellulase).  

The multi-component system according to the invention comprises at least one enzymatically active additive selected from the group of Hy enzyme class 3.2.1.

The hydrolase of enzyme class 3.2.1 is preferred around hemicellulases, e.g. B. xylanases, mannanases or cellulases.

The enzymatically active is particularly preferably Additive for an endo 1,4-β-xylanase (enzyme class 3.2.1.8) and / or an endo 1,4-β-glucanase (enzyme class 3.2.1.4).

The hydrolase of enzyme class 3.2.1 are commercially available or can be obtained using standard methods. The hydrolases of the Enzyme class 3.2.1 can either come from natural organisms be obtained or by means of known recombinant DNA techno produced and matched in appropriate expression systems be isolated from the culture medium according to the prior art. As organisms for the production of these enzymes come for example Plants, animal cells, bacteria and fungi are considered. Basically, both naturally occurring and gen technically modified organisms are enzyme producers. As well are parts of unicellular or multicellular organisms as En zym producers conceivable, especially cell cultures.

The hydrolases of enzyme class 3.2.1 can also be known molecular biological processes have been changed in a way be that they are suitable for the application described, for. B. by being opposite the activated mediator or a proteolytic activity were made insensitive.

The invention further relates to methods for delignifying materials containing lignin, which are characterized in that at least one oxidoreductase and at least one for the oxido reductase-suitable oxidizing agent and at least one  Mediator, which is an enzyme selected from the group of oxidors Ductases and hydrolases of enzyme class 3.2.1., not inactivated and at least one endohydrolase simultaneously or in any Sequence with an aqueous suspension of the lignin-containing Ma mixes terials.

Surprisingly, the process according to the invention succeeds the oxidative delignification system consisting of laccase and a mediator substance with a hydrolytic enzyme, such as. B. the xylanase and / or the cellulase in a single process step combine.

This is all the more surprising since it is known that with Verwen HBT as mediator the laccase itself in its activity is impaired (Amann, 1997) and simultaneously present En enzymes are irreversibly damaged and thus greatly reduced Show activity (Oksanen 1997).

The method according to the invention has the following advantages:

• - It improves the bleachability of pulp
• - It can be done easily with established chemical processes combine rens levels.

For example, also in combination with conventional process steps ten from pulp production, e.g. B. an alkaline extract tion, lignin can be selectively selected from the material used remove.

For example, ligninhal is used for the process according to the invention used material of plant origin, preferably which with the for the pulp or pulp position usual procedure has been unlocked.  

The material used can be unbleached pulp or mate be rial, which already with chemical delignification and Bleaching process has been treated.

In the process according to the invention, preferably 0.1-100 IU Oxidoreductase per g lignin-containing material (dry weight), particularly preferably 0.5-20 IU per g of lignin-containing material used.

The concentration of the oxidizing agent is preferably in the range range from 0.001 to 50 mmol / l. If as an oxidant What peroxide is used, so preferably in the range of 0.001-25 mmol / l.

The mediator is preferably used in amounts of
1-500 mmol / kg pulp, particularly preferably in amounts of
5-300 mmol / kg pulp, especially in amounts of
10-200 mmol / kg pulp (dry, absolutely dry).

Cellulases are preferably used in the process according to the invention and / or hemicellulases used as enzymatic additives. At for example, cellulase, xylanases or mannanases in pure Form or in the form of mixtures can be used. Xylanases who already used in the processing of pulp and by several manufacturers, e.g. B. Novo-Nordisk (Pulpzyme®), Clariant (Cartazyme®) offered. Cellulases are also commercially available true.

Depending on the conditions, which in the inventive method be set for the enzymatic delignification, correspondingly suitable variants (pH optimum, tempera stability) of these enzymes can be used.  

In the process according to the invention, the hydrolase becomes the enzyme class 3.2.1 in doses of 0.01-1000 IU enzyme / g pulp used, preferably in dosages of 1-500 IU enzyme / g, be particularly preferred in doses of 5-100 IU / g pulp (atro - absolutely dry).

Hemicellulases are preferably in doses of 0.1-1000 IU Enzyme / g pulp used, preferably in dosages of 1-500 IU enzyme / g, particularly preferably in doses of 5-100 IU / g Pulp (dry - absolutely dry) used.

Cellulases are preferably used in dosages of 0.01-500 IU Enzyme / g pulp used, preferably 0.05-100 IU enzyme / g Cellulose, particularly preferably 0.1-10 IU enzyme / g cellulose set.

The process according to the invention is usually carried out at temperature ren between 40 and 90 ° C, preferably between 45 and 70 ° C, particularly preferably carried out between 45 and 65 ° C.

Depending on the temperature stability of the enzymes used, too of which different optimal reaction conditions are set become. In general, good pH values can be found in the pH range from 3 to 10 Achieve delignification results, the is particularly preferred pH range from pH 4.5-7.

When using laccases there is sufficient availability of dissolved oxygen is a necessary condition. Depending on the ge The selected temperature can ensure the sufficient oxygen concentration by applying a sufficiently high pressure to the system to be achieved. The necessary partial pressure of oxygen can escape which is already guaranteed by the hydrostatic pressure or by applying a suitable pressure using a  suitable, oxygen-containing gas mixture, such as. B. air or also pure oxygen gas can be achieved.

The usual pressure range is in the range of an oxygen partial pressure (pO ₂ ) of 0.1-20 bar, preferably in the pressure range of 0.3-10 bar.

Depending on the choice of mediator, enzyme doses and reacti conditions, the process can expire at different speeds fen. The usual reaction time is between 30 min. and 2 hours.

The pulp concentration (consistency) of the invention The process is usually in the range of 1-25%, preferred in the range 6-20%, particularly preferably in the range 10-15%.

In the following examples, the invention will be further wrote.

The following components are used in the examples:

Mediators:
Mediators were obtained from the companies Aldrich, Merck or Janssen or were produced using customary and generally known methods. Some mediators are given abbreviations in the text as follows: 1-OH-benzotriazole (HBT), violuric acid (Vio), N-OH-acetanilide (NHA).

Enzymes:
Laccase:
Laccase from Trametes versicolor obtained by fermentation was used.

Activity determination:
Laccase activity is determined via the oxidation of 2,2'-azino-bis- (3-ethylbenzothiazoline-6-sulfonate), ABTS, Boehringer Mannheim, under aerobic conditions. The green color resulting from the enzymatic reaction is measured photometrically at 420 nm. The reaction temperature is 25 ° C, the pH 4.5. A laccase unit is the amount of enzyme that catalyzes the conversion of 1 mol ABTS / min under the specified conditions. An extinction coefficient of 36,000 M⁻ ¹ cm⁻ ^{1 is} used to calculate the turnover.

Cellulase:
Commercially available cellulases from Fluka (Trichoderma viride), Sigma (Aspergillus niger, Trichoderma viride, Penicillium funiculosum), Röhm (Rohalase 7069) were used. The dosage was according to the activity information of the manufacturer.

Xylanase:
Commercially available xylanases from Fluka (Trichoderma viride) or Clariant were used. The dosage was made according to the activity information of the manufacturers.

Pulp:
Hardwood (HW) and Softwood (SW) Kraft pulp were used.
Softwood: Kappa 14.5 (after extraction)
Hardwood: Kappa 12 (after extraction).

The delignification was assessed by determining the Kappa value (according to TAPPI method T236) of the treated pulp sample after alkaline extraction. The alkaline extraction (E stage) was carried out at 2% consistency, 60 ° C with 80 g NaOH / kg cell fabric and a duration of 60 min.  

example 1 Comparison of the delignification of a SW pulp with one Laccase-Mediator System (LMS®) with simultaneous presence / ab presence of xylanase

The following was tabulated in a 500 ml laboratory autoclave described approach for two hours at 45 ° C (water bath) under Application of an oxygen partial pressure of 10 bar incubated (L level).

L level

5 g softwood pulp (dry)
Fabric density: 10%
Laccase: 5 IU / g pulp
Mediator: N-OH-Acetanilide: 9 mg / g pulp
pH: 4.5
Total liquid volume: 50 ml.

To the positive effect of an enzymatic additive such. B. the xylanase, was determined to be the L level described as further additive Xylanase from Trichoderma viride (Fluka) added (XL level), so that the approach was composed as follows:

(XL) level

5 g softwood pulp (dry)
Fabric density: 10%
Laccase: 5 IU / g pulp
Xylanase: 50 IU / g pulp
Mediator: N-OH-Acetanilide: 9 mg / g pulp
pH: 4.5
Total liquid volume: 50 ml.

Both approaches were then 60 min. long alkaline extra here (E level).  

E level

2% fabric density
Temperature: 60 ° C
Alkali: 80 g NaOH / kg pulp.

The degree of delignifi was determined from the determination of the kappa values ornamentation determined. The control value gives the kappa value of the off starting pulp after alkaline extraction.

Table 1

Trial evaluation

The addition of xylanase increases delignification by approx. 30%.

Example 2 Comparison of delignification of a HW pulp with a lac case-mediator system (LMS®) with simultaneous presence / deviation xylanase

The example corresponds to example 1, with the difference that a hardwood pulp was used instead of softwood. The following was tabulated in a 500 ml laboratory autoclave described approach for two hours at 45 ° C (water bath) under Application of an oxygen partial pressure of 10 bar incubated (L level).  

L level

5 g hardwood pulp (dry)
Fabric density: 10%
Laccase: 5 IU / g pulp
Mediator: N-OH-Acetanilide: 9 mg / g pulp
pH: 4.5
Total liquid volume: 50 ml.

To the positive effect of an enzymatic additive such. B. the xylanase, was determined to be the L level described as further additive Xylanase from Trichoderma viride (Fluka) added (XL-Sufe), so that the approach was composed as follows:

(XL) level

5 g hardwood pulp (dry)
Fabric density: 10%
Laccase: 5 IU / g pulp
Xylanase: 50 IU / g pulp
Mediator: N-OH-Acetanilide: 9 mg / g pulp
pH: 4.5
Total liquid volume: 50 ml.

The batches were then 60 min. extracted long alkaline (E level).

E level

2% fabric density
Temperature: 60 ° C
Alkali: 80 g NaOH / kg pulp.

The degree of delignifi was determined from the determination of the kappa values ornamentation determined. The control value gives the kappa value of the off starting pulp after alkaline extraction.  

Table 2

Trial evaluation

The addition of xylanase increases delignification by approx. 96%.

Example 3 Influence of different xylanases on the delignification of a Pulp with a laccase mediator system (LMS®)

In order to investigate the effects of the addition of different xylanases, the delignification effect was investigated in accordance with the (XL) step described in Example 1 when using different xylanases. The following were used as xylanases:
Clariant: Cartazym HS 10, liquid
Fluka: Trichoderma viride xylanase.

L and (XL) stages were carried out according to Example 1, each however, the batches were run at pH 6.0. The xylanose dose tion was 12 IU / g pulp, laccase was 15 IU / g Dosed pulp.

The batches were then 60 min. extracted long alkaline (E stage, example 1). From the determination of the kappa values the degree of delignification is determined.  

Table 3

Results

In order to achieve optimal delignification, the respective best combinations of pulp, LMS and xylanase determined become. Different xylanases lead to, as the example shows differently good delignifications.

However, all results are better than without xylanase.

Example 4 Mediator dependence of delignification with a laccase me diator system (LMS®) with simultaneous presence of xylanase

To determine the influence of the mediator on the system, the delignification effect of those described in Example 1 (XL) level when using different mediators under is looking for.

The following were used as mediators (abbreviations in brackets):
1-OH-benzotriazole (HBT), violuric acid (Vio), N-OH-acetanilide (NHA), N-OH-phthalimide (HPI), 1-nitroso-2-naphthol-3,6-disulfonic acid disodium salt hydrate (1- NNS), 2-nitroso-1-naphthol-4-sulfonic acid tetrahydrate (2-NNS).

The batches were carried out as in Example 1. In detail did they contain:  

(XL) level (mediator dependency)

5 g softwood pulp (dry)
Fabric density: 10%
Laccase: 15 IU / g pulp
Xylanase (Trichoderma): 25 IU / g pulp
Mediators: 75 mmol / kg pulp
pH: 6.0
Total liquid volume: 50 ml.

The batches were then 60 min. extracted long alkaline (E stage) as described in Example 1.

The degree of delignifi was determined from the determination of the kappa values ornamentation (Delig.) determined. The control value gives the kappa value of the original pulp after alkaline extraction.  

Table 4

Trial evaluation

The xylanase effect depends heavily on the mediator used. Wuh delignification when using 1-OH-benzotriazole (HBT) almost does not increase (+ 5%) significant increases are observed with other mediators den (e.g. NHA + 17.8%).

Example 5 pH dependence of the delignification of a pulp with a Laccase Mediator System (LMS®) in the presence of Xylanase

To determine the influence of pH on the system, the delignification effect of those described in Example 1  (XL) level examined at different pH values. As ver Appropriate approaches without xyl 20236 00070 552 001000280000000200012000285912012500040 0002019804583 00004 20117anase (L stage) served the same purpose. The Batches were carried out as in Example 1. In detail ent did they hold:

(XL) level- (pH dependence)

5 g softwood pulp (atro) kappa 14.5
Fabric density: 10%
Laccase: 15 IU / g pulp
Xylanase (Trichoderma viride): 25 IU / g pulp
Mediator: N-OH-Acetanilide: 9 mg / g pulp
pH values: 4/5/6/7 (with McIllvaine buffer)
Total liquid volume: 50 ml.

Both approaches were then 60 min. long alkaline extra here (E level).

The degree of delignifi was determined from the determination of the kappa values ornamentation determined.

Table 5

Trial evaluation

By adding xylanase to the laccase mediator system a drastic improvement in performance can be achieved. Of the Effect shows a clear pH dependence. At all pH values  However, the addition of xylanase is an improvement pass.

Example 6 Dependency of the delignification of a pulp with a lac case mediator system (LMS®) with simultaneous presence at different amounts of xylanase (dose-dependent)

To determine the influence of the xylanase dosage on the system men, the delignification effect of the be in Example 1 written (XL) level at different xylanase doses examined. A corresponding approach without served as a comparison Xylanase (L level). The batches were carried out as in Example 1 leads. In detail they contained:

L level (reference value without xylanase)

5 g softwood pulp (dry)
Fabric density: 10%
Laccase: 5 IU / g pulp
Mediator: N-OH-Acetanilide: 9 mg / g pulp
pH: 4.5
Total liquid volume: 50 ml.

(XL) -stage- (various amounts of xylanase)

5 g softwood pulp (atro) kappa 14.5
Fabric density: 10%
Laccase: 5 IU / g pulp
Xylanase (Trichoderma viride): 12.5 / 25 / 37.5 / 50/100/200 IU / g pulp
Mediator: N-OH-Acetanilide: 11 mg / g pulp
pH: 4.5
Total liquid volume: 50 ml.

After the incubation, the batches were 60 min. long alkaline ex trailed (E-level).

The degree of delignifi was determined from the determination of the kappa values ornamentation determined.

Table 6

Trial evaluation

Improving the effectiveness of the laccase mediator system depends on the dose of xylanase added.

Example 7 Delignification of a SW pulp with a laccase mediator System (LMS®) which contains cellulase as an additive

The following was tabulated in a 500 ml laboratory autoclave described reference approach for 2 hours at 45 ° C (water bath) incubated applying an oxygen partial pressure of 10 bar (L level).  

L level (comparison test)

5 g softwood pulp (dry)
Fabric density: 10%
Laccase: 15 IU / g pulp
Mediator: N-OH-Acetanilide: 10 mg / g pulp
pH: 4.5
Total liquid volume: 50 ml.

To the positive effect of an enzymatic additive such. B. the cellulase, to be determined, was one of the L level the approach as a further additive cellulase from Trichoderma viride (Sigma) added (CL-Sufe), so that the approach together as follows was set:

(CL) level

5 g softwood pulp (dry)
Fabric density: 10%
Laccase: 15 IU / g pulp
Cellulase: 10 U / g pulp
Mediator: N-OH-Acetanilide: 10 mg / g pulp
pH: 4.5
Total liquid volume: 50 ml.

Both batches were then made alkaline extra for 60 minutes here (E level).

E level

2% fabric density
Temperature: 60 ° C
Alkali: 80 g NaOH / kg pulp.

The degree was determined from the determination of the kappa values of both approaches of delignification. The control value gives the Kappa value of the original pulp after alkaline extraction.

Table 7

Trial evaluation

The addition of cellulase increases the delignification of one Softwood pulp versus a cellulase free reference by around 43%.

Example 8 Delignification of a HW pulp with a laccase mediator System (LMS®) which contains cellulase as an additive

The following was tabulated in a 500 ml laboratory autoclave described reference approach for 2 hours at 45 ° C (water bath) incubated applying an oxygen partial pressure of 10 bar (L level).

L level (comparison test)

5 g hardwood pulp (dry)
Fabric density: 10%
Laccase: 15 IU / g pulp
Mediator: N-OH-Acetanilide: 10 mg / g pulp
pH: 4.5
Total liquid volume: 50 ml.

To the positive effect of an enzymatic additive such. B. the cellulase to be determined was one of the L level Approach as a further additive cellulase from Trichoderma vi ride (Sigma) added (CL-Sufe), so the approach is as follows was composed:

(CL) level

5 g hardwood pulp (dry)
Fabric density: 10%
Laccase: 15 IU / g pulp
Cellulase: 10 U / g pulp
Mediator: N-OH-Acetanilide: 10 mg / g pulp
pH: 4.5
Total liquid volume: 50 ml.

Both approaches were then 60 min. long alkaline extra here (E level).

E level

2% fabric density
Temperature: 60 ° C
Alkali: 80 g NaOH / kg pulp.

The degree was determined from the determination of the kappa values of both approaches of delignification. The control value gives the Kappa value of the original pulp after alkaline extraction.  

Table 8

Trial evaluation

The addition of cellulase increases the delignification of one Hardwood pulp versus a cellulase-free reference 108%.

Example 9 Influencing cellulase activity by the laccase mediator System (LMS®) when using different mediators

An effect on the delignifiability of pulp can only then be observed when the cellulase is present in the presence of the Lac case-mediator system is enzymatically active. The activity of Cellulases were therefore in a cellulose-free system, which Contained laccase and mediator, measured. Zellu was examined Read from Aspergillus niger and Trichoderma viride. As mediators 1-OH-benzotriazole (HBT), violuric acid (Vio) and N-OH-Ace tanilide (NHA) used.

The cellulase activity was measured in an optical Test with dinitrosalicylic acid (DNSA) as an oxidizing agent such as described below.

approaches

The batches contained 20 mg in a total volume of 10 ml Cellulase, 0.8 mg laccase and 75 µmol mediator at pH  of 6.2. The approach was 50 µl after different times removed and the cellulase activity determined

Cellulase test

Principle: determining the amount of reducing sugar which by enzymatic hydrolysis from carboxymethyl cellulose (CMC) is released. A downstream redox reaction between to set DNSA and the enzymatically formed reducing Sugar ends leads to a coloration of the solution, which is photometric can be quantified at 595 nm.

For the enzyme test 325 µl test buffer (0.1 mol / l citrate / phosphate pH 6.2 with 0.4 mol / l NaCl), 125 µl CMC solution (2% in test buffer; Na salt) and 50 µl of pipetted solution to be tested pipetted together and incubated at 45 ° C for different lengths. At the end of the incubation, 750 µl DNSA solution (1 g 3,5-dinitrosalicylic acid, 0.2 g phenol, 0.05 g Na ₂ SO ₃ and 20 g NaK tartrate are dissolved in 100 ml 1% NaOH). added ben and the mixture then heated at 95 ° C for 20 min. The solution is cooled on ice, any precipitated material is centrifuged off (2 min, 14,000 rpm, Eppendorf centrifuge) and the absorbance in the supernatant is determined at 595 nm. The formation of reducing sugars can be quantified by comparison with a calibration curve (glucose). 1 unit (1U) is the enzyme activity which releases 1 µmol of glucose under the specified conditions from CMC cellulose in one minute.

Table 9

Time dependence of the cellulase activity of Aspergillus niger in the presence of the laccase-mediator system when using various mediators (HBT, NHA, Vio). The numbers indicate the relative cellulase activity as a percentage of the initial value (rel.%).

Table 10

Time dependence of the cellulase activity of Trichoderma viride in the presence of the laccase-mediator system when using different mediators (HBT, NHA, Vio). The numbers indicate the relative cellulase activity as a percentage of the initial value (rel.%).

The enzymatic activity of the A. niger and T. viride takes with simultaneous incubation in the laccase mediator System. The slightest loss can be found with NHA as media gate. A dependency on the enzyme used is also to be take care.

Example 10 Delignification of a SW pulp with a laccase mediator System (LMS®) which as an additive cellulase different Origin contains

The following was tabulated in a 500 ml laboratory autoclave described reference approach for 2 hours at 45 ° C (water bath) incubated applying an oxygen partial pressure of 10 bar (L level).

L level (comparison test)

5 g softwood pulp (dry)
Fabric density: 10%
Laccase: 15 IU / g pulp
Mediator: N-OH-Acetanilide: 10 mg / g pulp
pH value: depending on cellulase pH 5-6 (table)
Total liquid volume: 50 ml.

To the positive effect of an enzymatic additive such. B. the cellulase to be determined was one of the L level Appropriate approach as a further addition of different cellulase added (CL-Sufe), so that the approach is composed as follows was:

(CL) level

5 g softwood pulp (dry)
Fabric density: 10%
Laccase: 15 IU / g pulp
Cellulase (various origins): 10 U / g pulp
Mediator: N-OH-Acetanilide: 10 mg / g pulp
pH value: depending on cellulase pH 5-6 (Table 11)
Total liquid volume: 50 ml.

Both batches were then made alkaline extra for 60 minutes here (E level).

E level

2% fabric density
Temperature: 60 ° C
Alkali: 80 g NaOH / kg pulp.

The degree was determined from the determination of the kappa values of both approaches of delignification. The control value gives the Kappa value of the original pulp after alkaline extraction.

Table 11 Trial evaluation

Improvement of the delignification with the Laccase-Mediator system with simultaneous presence of cellulases from different origins.

With all cellulase used, the delignification performance can of the Laccase mediator system can be improved.

Example 11 Influence of the cellulase dose on delignification using of a laccase mediator system (LMS®) which as an additive cell lase contains

The following was tabulated in a 500 ml laboratory autoclave described reference approach for 2 hours at 45 ° C (water bath) incubated applying an oxygen partial pressure of 10 bar (L level)

L level (comparison test)

5 g softwood pulp (dry)
Fabric density: 10%
Laccase: 15 IU / g pulp
Mediator: N-OH-Acetanilide: 10 mg / g pulp
pH: pH 6
Total liquid volume: 50 ml.

To the positive effect of the enzymatic additive (cellulase) too was determined as an approach corresponding to the L level further addition different amounts of cellulase added (CL level), so that the approach was composed as follows:

(CL) level

5 g softwood pulp (dry)
Fabric density: 10%
Laccase: 15 IU / g pulp
Cellulase (Trichoderma viride-Sigma) 0-30 U / g pulp
Mediator: N-OH-Acetanilide: 10 mg / g pulp
pH 6
Total liquid volume: 50 ml.

Both batches were then made alkaline extra for 60 minutes here (E level).  

E level

2% fabric density
Temperature: 60 ° C
Alkali: 80 g NaOH / kg pulp.

The degree was determined from the determination of the kappa values of both approaches of delignification. The control value gives the Kappa value of the original pulp after alkaline extraction. Around the influence of the combined treatment on cellulose The viscosity of the pulp samples was examined. The viscosity is determined in accordance with the regulations according to TAPPI. The viscosity is given in ml / g.

Table 12 Trial evaluation

Influence of the cellulase dose on delignification and viscosity of pulp (SW; K 16.6) when using a cellulase-containing sequence (CL) E.

By increasing the cellulase dose you can go up to a range  of 9 U / g pulp an improvement in delignifiability achieve. If the dose is increased further, there will be no improvement achieved more. The viscosity of the pulp remains up to one Dosage of approx. 3 U / g almost constant. With further dose increases there is a significant loss of viscosity. At approve Neter dosage (3 U / g in the example), the positive cell u Use the lase effect to improve delignification without damage to the fiber (viscosity drop) occurs.

Example 12 Influence of pH on delignification using a Laccase-Mediator Systems (LMS®) which as an additive cellulase contains

In order to demonstrate the influence of the pH value, the delignifi approach compared at different pH values. In a 500 ml laboratory autoclave was subsequently tabulated Written approach for 2 hours at 45 ° C (water bath) under An incubate an oxygen partial pressure of 10 bar (CL level).

(CL) level

5 g softwood pulp (dry)
Fabric density: 10%
Laccase: 15 IU / g pulp
Trichoderma viride (Sigma) cellulase: 2.5 U / g pulp
Mediator: N-OH-Acetanilide: 10 mg / g pulp
pH values: 4, 5, 6, 7, 8
Total liquid volume: 50 ml.

After the incubation, the batches were alkaline ex for 60 min trailed (E-level).  

E level

2% fabric density
Temperature: 60 ° C
Alkali: 80 g NaOH / kg pulp.

From the determination of the kappa values of the deligni in this system the degree of delignification Right.

Table 13

pH dependence of the delignification with a laccase mediator system, which contains cellulase as an additive (cellulose has Kappa 16.6 according to E level)

Table 13 shows that the laccase mediator system, which contains cellulase as an additive, with a clear pH profile Optimal for the selected components in the range of pH 6 in the Shows delignification.

Example 13 Influence of the mediator on delignification using a Laccase-Mediator Systems (LMS®) which as an additive cellulase contains

The following were tabulated in 500 ml laboratory autoclaves  described reference approaches for 2 hours at 45 ° C (water bath) incubated applying an oxygen partial pressure of 10 bar (L level).

L level (comparison test)

5 g softwood pulp (dry)
Fabric density: 10%
Laccase: 15 IU / g pulp
Mediator: N-OH-Acetanilide: 10 mg / g pulp
pH: 6.0
Total liquid volume: 50 ml.

To the positive effect of an enzymatic additive such. B. the cellulase to be determined was one of the L level Approach as a further additive cellulase from Trichoderma vi ride (Sigma) added (CL-Sufe), so the approach is as follows was composed:

(CL) level

5 g softwood pulp (dry)
Fabric density: 10%
Laccase: 15 IU / g pulp
Cellulase: 2.5 U / g pulp
Mediator: N-OH-Acetanilide: 10 mg / g pulp
pH: 6.0
Total liquid volume: 50 ml.

Both batches were then made alkaline extra for 60 minutes here (E level).

E level

2% fabric density
Temperature: 60 ° C
Alkali: 80 g NaOH / kg pulp.

The degree of delignifi was determined from the determination of the kappa values ornamentation determined. The starting pulp had a kappa value of 16.6 after alkaline extraction.

Table 14

Delignification (rel.%) With the Laccase Mediator System, which contains cellulase as an additive compared to a cellulase-free system.

The addition of cellulase increases the delignification of one Softwood pulp versus a cellulase free reference in Presence of all mediators used.

Example 14 Delignification of various pulps with a Laccase-Me diator system (LMS®) which is an additive for cellulase and xylanase contains

In 500 ml laboratory autoclaves were tabulated below Written reference approaches for 2 hours at 45 ° C (water bath) incubated applying an oxygen partial pressure of 10 bar (L level).

L level (comparison test)

5 g different cellulose (dry)
Fabric density: 10%
Laccase: 15 IU / g pulp
Mediator: N-OH-Acetanilide: 10 mg / g pulp
pH: 6.0
Total liquid volume: 50 ml.

To the positive effect of several simultaneous enzymatic Ad ditives such as B. the cellulase and xylanase to determine approaches corresponding to the L level were added Trichoderma viride (Sigma) cellulase and xylanase added (CLX-Sufe), so that the approaches were composed as follows:

(LXC) level

5 g different cellulose (dry)
Fabric density: 10%
Laccase: 15 IU / g pulp
Cellulase: 0.1 U / g pulp
Xylanase: 2.0 U / g
Mediator: N-OH-Acetanilide: 10 mg / g pulp
pH: 6.0
Total liquid volume: 50 ml.

After the incubation, all batches were alkaline ex for 60 min trailed (E-level).

E level

2% fabric density
Temperature: 60 ° C
Alkali: 80 g NaOH / kg pulp.

The degree of delignifi was determined from the determination of the kappa values ornamentation determined. The achievable Delignifi were compared Degree of ornamentation (rel.%) of treatment with laccase / mediator (LE) (comparative experiment) with treatment with Laccase / Me  diator / cellulase / xylanase (LXC) E.

Table 15

Comparison of delignifications of a laccase mediator system - sequence (LXC) E - provided with cellulase and xylanase additive with the additive-free system - sequence LE -. Different pulps were tested.

With all pulps there can be a significant increase in deligni fication by means of the Laccase Mediator System by the simultaneous gene addition of cellulase and xylanase can be achieved.

Claims (10)

1. Multi-component system for changing, breaking down or bleaching lignin, lignin-containing materials or similar substances, containing an oxidoreductase and an oxidizing agent suitable for the oxidoreductase and a mediator and at least one enzymatically active additive, characterized in that the mediator has the oxidoreductase and the enzymatically active same additive not inactivated and the enzymatically active additive from the group of hydrolases of the enzyme class 3.2.1. is selected. 2. Multi-component system according to claim 1, characterized net that the mediator from the group of aliphatic, cy cloaliphatic, heterocyclic or aromatic compound is selected that contains at least one N-Hxdroxy-, Oxim-, Contain nitroso, N-oxyl or N-oxi function, sub substituted or unsubstituted 1-hydroxy-1-benzotriazoles, 3H-Benzotriazole-1-oxide and 2H-Benzotriazole-1-oxide except men are. 3. Multi-component system according to claim 1, characterized net that the mediator from the group of compounds according to Formulas I to XXXIV of the description is selected. 4. Multi-component system according to claim 1 to 3, characterized records that the enzymatically active additive from the group of xylanases, mannanases and cellulases is selected. 5. Multi-component system according to claim 4, characterized net that the enzymatically active additive is egg ne Endo 1,4-β-xylanase (enzyme class 3.2.1.8) and / or Endo 1,4-β-glucanase (enzyme class 3.2.1.4). 6. Multi-component system according to one or more of the claims 1 to 5, characterized in that as an oxidoreductase Laccase is used. 7. Process for delignification of lignin-containing materials s, which are characterized in that at least one  Oxidoreductase and at least one for the oxidoreductase suitable oxidizing agent and at least one mediator Enzyme selected from the group of oxidoreductases and Hy Drolases of enzyme class 3.2.1., not inactivated and min at least one endohydrolase simultaneously or in any order sequence with an aqueous suspension of the lignin-containing Material mixes. 8. The method according to claim 7, characterized in that 0.1-100 IU oxidoreductase per g lignin-containing material (dry weight) can be used. 9. The method according to claim 7 or 8, characterized in that the mediator in amounts of 1-500 mmol / kg pulp is set. 10. The method according to claim 7, characterized in that the Enzyme class hydrolase 3.2.1. in doses of 0.01-1000 IU Enzyme / g of pulp is used.