US20060054290A1

US20060054290A1 - Novel catalytic activities of oxidoreductases for oxidation and or bleaching

Info

Publication number: US20060054290A1
Application number: US10/508,184
Authority: US
Inventors: Hans-Peter Call
Original assignee: Individual
Current assignee: Individual
Priority date: 2002-01-26
Filing date: 2003-01-26
Publication date: 2006-03-16
Also published as: WO2003061550A2; CA2514475A1; EP1483442A2; DE10203135A1; AU2003206637A1; WO2003061550A3

Abstract

A new enzyme-based process for oxidation and/or bleaching is described, comprising of oxidoreductases such as laccases and/or peroxidases—seperately or in combination—in the presence of their respective co-substrates like O₂, air, H₂O₂, organic peroxides, peracids etc. and comprising of enhancer compounds from the class of oxocarbons, from the class of urazoles and hydrazides, from the class of hydantoins and the class of nitril (Cyan)-compounds, and comprising additionally of carbonyl compounds such as ketones, aldehyds, whereby the combination of enzyme, co-substrate, enhancer compound and carbonyl compound generate active oxygen species like dioxiranes, dioxetanes, peroxy-compounds etc. or form other reactive compounds or transition states like radicals (kation radicals, anion radicals) or reactive (red/ox-active) neutral compounds as oxidizing and/or bleaching agents.

Description

It is wellknown, that enzymes like e.g. peroxidases, especially horseradish peroxidase (HRP), lignin peroxidases and manganese peroxidases and also special oxidases such as laccases, which all belong to the lignolytic enzymes, can oxidize a huge variety of different substrates either in the presence of H₂O₂, organic peroxides or peracids (peroxidases) or in the presence of air or O₂(oxidases/laccases).
During the last years several applications of the mentioned enzymes were published or patented like e.g. their use as mild oxidants in the chemical synthesis or in food field, as decolorizing oxidants of dyes e.g. in waste waters, as labeling-enzymes for special analysis approaches and in biosensors etc.
Furthermore some applications were published and patented referring to enzymes which work together with special mediator compounds (WO96/18770/, WO 94/29510, WO 94/12619, WO 94/12620 und WO 94/12621).
In these cases the preferred enzyme is mostly laccase, an enzyme which is today commercial available.
On the one hand these mediator compounds make possible an oxidation of substrates with higher oxidation potential, they normally cannot be oxidized by laccases alone; and on the other hand they cause that the so-called laccase mediator system—due to the fact that the mediator compounds are free-diffusible and low-molecular—can also degradate substrates in not freely accessable substances such as e.g. lignin in pulp fibres. Free enzymes wouldn't cause any significant substrate degradation due to their—besides the low oxidation power—too large molecule size.
Besides these laccase-based enzymatic approaches only the manganes peroxidase systems (MnP-systems) which can be found in withe-rotting fungi and work with the aid of chelated Mn²⁺ as natural mediator can be applied as an in-vivo-system for lignin degradation in pulps. The main drawbacks regarding the performance of these systems are on the one hand their strong sensitivity against H₂O₂as co-oxidant and on the other hand the difficulties in production of the enzymes. These difficulties have made a use in a larger scale and therefore a commercialization impossible.
Due to the too large molecular weight which makes a penetration into the fibres impossible (see above) und due to the fact that there are so far no specific mediator compounds have Due to the too large molecular weight which makes a penetration into the fibres impossible (see above) und due to the fact that there are so far no specific mediator compounds have been found, the use of lignin peroxidase causes no delignification but a lignin polymerisation.
Despite the good performance of laccase- mediator-systems (LMS) and the very good selectivity i.e. good pulp properties which can be obtained, a) the high price mainly of the mediator compounds b) their necessary relatively high dosages (the reactions are stochiometric and not catalytic) and c) the oftenly existing environmental risks due to the possible toxicity of the mediator compounds prevent the broad implementation of these systems.
On the other side there is a great demand for alternative bleaching systems mainly due to the necessity for closed mill loops which can only difficult be obtained (because of e.g. of the corrosion problems) with the aid of ECF-bleaching (elemental chlorine free bleaching) with chlorine dioxide as main bleaching agent.
Therefore it is the target of the present invention to provide an oxidation-/bleaching system which hasn't the disadvantages of the mentioned state-of-the-art systems.
This can be solved by providing a new process for oxidation and/or bleaching comprising of

ACCORDING TO THE INVENTION

oxidoreductases such as laccases and/or peroxidases—seperately or in combination—in the presence of their respective co-substrates like O₂, air, H₂O₂, organic peroxides, peracids etc. and comprising of
enhancer compounds from the class of oxocarbons, from the class of urazoles and hydrazides, from the class of hydantoins and the class of nitril (Cyan)-compounds, and comprising additionally of
carbonyl compounds such as ketones, aldehyds,
whereby the combination of enzyme, co-substrate, enhancer compound and carbonyl compound generate active oxygen species like dioxiranes, dioxetanes, peroxy-compounds etc. or form (according e.g. to the literature: K. Deuchert et. al., Angewandte Chemie 90, S. 927-938, 1978) also other reactive compounds or transition states like radicals (kation radicals, anion radicals) or reactive (red/ox-active) neutreal compounds as oxidizing and/or bleaching agents.
As oxocarbon compounds such compounds are preferably used as cited in the literature: Chemie in unserer Zeit, 16. Jahrgang 1982, Nr. 2, S. 57-67 inclusivly of the therein quoted literature preferably: R.West et al., Oxocarbons and their Reactions, in J. Zabicky ed., “The chemistry of the Carbonyl Group”, Wyley (Interscience), 1970; R.West, Oxocarbons,
Academic
Press, 1980 und Römpp Chemie Lexikon, Thieme Verlag, 1995, S. 3175-3177.
Particularly preferred are carbonyl compounds of the general formula I, such as α-hydroxycarbonyl compounds of general formula Ia, α-dicarbonyl compounds of general formula lb, β-hydroxycarbonyl compounds of general formula Ic and β-dicarbonyl compounds of general formula Id:

wherein R¹to R⁸independently of one another are selected from the group consisting of hydrogen, a halogen, an alkyl group, an alkyloxy group, an aryl group, an aryloxy group, a hydroxy group, an oxo group, a formyl group, a thioxo group, a mercapto group, an alkylthio group, a sulfeno group, a sulfino group, a sulfo group, a sulfamoyl group, an amino group, an imino group, an amido group, an amidino group, a hydroxycarbamoyl group, a hydroximino group, a nitroso group, a nitro group and a hydrazono group, with R¹and R²; R³and R⁴; R⁵and R⁶; and R⁷and R⁸possibly being linked together to form a single group, and wherein n is greater than or equal to 1;
particularly preferred are such compounds of general formula II (linear compounds with double bonds/enols):

wherein R⁹and R¹⁰, independently of one another, are selected from the group consisting of hydrogen, a halogen, an alkyl group, an alkyloxy group, an aryl group, an aryloxy group, a hydroxy group, an oxo group, a formyl group, a thioxo group, a mercapto group, an alkylthio group, a sulfeno group, a sulfino group, a sulfo group, a sulfamoyl group, an amino group, an imino group, an amido group, an amidino group, a hydroxycarbamoyl group, a hydroximino group, a nitroso group, a nitro group and a hydrazono group, and R⁹and R¹⁰are possibly linked together to form a single group;
Furthermore preferred are such compounds of general structure III (cyclic compounds, groups not OH, derivatives of squaric acid, OH group derivatized):

wherein R¹¹and R¹², independently of one another are selected from the group consisting of hydrogen, a halogen, an alkyl group, an alkyloxy group, an aryl group, an aryloxy group, a hydroxy group, an oxo group, a formyl group, a thioxo group, a mercapto group, an alkylthio group, a sulfeno group, a sulfino group, a sulfo group, a sulfamoyl group, an amino group, an imino group, an amido group, an amidino group, a hydroxycarbamoyl group, a hydroximino group, a nitroso group, a nitro group and a hydrazono group, and m is greater than or equal to 0. Particularly preferred are cyclic oxocarbon compounds of the general formula IV (general sum formula: C_xO_xwherin x=>3.
Struktur element:
p≧0.
Particularly preferred are compounds such as, for example: deltic acid, squaric acid, croconic acid and rhodizonic acid.
Particularly preferred are also tetrahydroxy-p-benzoquinones their saltse and derivatives and the corresponding salts and derivatives of the deltic acid, squaric acid, croconic acid and rhodizonic acid
Also particularly prteferred are derivatives of squaric acid according to the literature: K. Deuchert et. al., Angewandte Chemie, 90, 1978, S. 927-938.
Particularly preferred compounds from the group of amides are hydrazides and urozoles of the general formula V (amides) and VI (hydrazides):

wherein X is C═O or O═S═O; R is the same or different and independently of one another is hydrogen, an alkyl group, an aryl group or an acyl group; wherein X is C═O or O═S═O; R is the same or different and independently of one another is hydrogen, and alkyl group, an aryl group or an acyl group;
Most particularly preferred are compounds like carbazates such as methyl-, ethyl-, tert, butyl-, benzylcarbazate etc. and pyrazoles and respective derivatives.
Particularly preferred are cyclic hydrazides of the general formula VII

wherein X is C═O or O═S═O (cyclic hydrazides of dicarbonic acids or disulfonic acids).
R is the same or different and independently of one another is hydrogen, an alkyl group, an aryl group or an acyl group.
G is selected from the group consisting of CH₂, CH₂—CH₂, CHR¹—CHR¹, CH═CH, CR₂—CR₂, NH, NR³, C═O, ortho-C₆H_4,ortho-C₁₀H₆, wherein R¹to R³are the same or different and independently of one another are hydrogen, an alkyl group, an aryl group, or an acyl group;
Furthermore preferred are urazoles (formula VIII) und phthalhydrazides (Formula IX)

wherein R⁴is hydrogen, an alkyl group, an alkoxy group, a carboxyl group, a nitro group or an amino group;
R is the same or different and independently of one another is hydrogen, an alkyl group or an aryl group.
Particularly preferred are compounds such as: maleic hydrazide, 2-nitrobenzhydrazide, p-toluenesulfonyl hydrazide, nicotinic hydrazide, isonicotinic hydrazide, 4,4′-oxydibenzenesulfonyl hydrazide, benzoichydrazide, phthalic hydrazide, 3-aminophthalic hydrazide, 1-naphthoic hydrazide, 3-hydroxy-2-naphthoic hydrazide, hydroxybenzhydrazide, oxamic hydrazide, oxalic dihydrazide, terephthalic dihydrazide, isophthalic dihydrazide, L-tyrosine hydrazide, oxalic bis-(benzylidene hydrazide), salicylidenesalicylic hydrazide, thiophene 2-carbonic acid hydrazide, furan 2-carbonic acid hydrazide.
Furthermore particularly preferred are 5-arnino-S-hydroxypyrazole, 2,3-dihydrophthalazine-1,4-dione, 7-nitroindazole, 1,2-dihydropyrazine-3,6-dione.
Also particularly preferred are 4-phenylurazole, 1-phenylurazole, 4-methylurazole, 4-tert, -butyluralzole and urazole.
From the group of imides as e.g. hydantoins, particularly compounds of the general formula X (imides) are used:

wherein the R is the same or different and independently of one another is hydrogen, an alkyl group, an aryl group, an acyl group or an amino group.
Also particularly preferred are imides of thegeneral formula XI:

wherein the R is the same or different and independently of one another is hydrogen, an alkyl group, an aryl group, an acyl group or an amino group.
Particularly preferred are cyclische Imide of the general formula XII:

R is the same or different and independently of one another is hydrogen, an alkyl group, an aryl group, an acyl group or an amino group; G is selected from the group consisting of CH₂, CHR¹, CR¹R², CH═CH, CR³═CR⁴, NH, NR⁵, C═O, and O, wherein R¹to R⁵are the same or different and independently of one another are hydrogen, an alkyl group, an aryl group, an alkoxy group, or a carboxy group.
Furthrermore particularly preferred are derivatives of hydantoins (formula XIII), like compounds, wherein the hydantoin derivative is selected from the group consisting of diethyl-5-hydantoyl phosphonate, 5-methyl-5-phenylhydantoin, hydantoyl-5-acetic acid and 1,3-dibromo-5,5-dimethylhydantoin.
As nitrile compounds such compounds according to the literature: Römpp Chemie
Lexikon, Thieme Verlag, 1995, S. 3012-3013 and the herein cited literature e.g.: Chem. Unserer Zeit, 18, 1984,S.1-16 are used.
Particularly preferred are cyanamide and dicyandiamide.
Enzymes
Oxidoreductases are preferrably used as enzymes. Particularly preferred enzymes are laccases and peroxidases or genereally oxidoreductases of the classes 1.1.1. to 1.97 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) among which the following are particularly preferred:
enzymes of the class 1.1, particularly preferred of the class 1.1.5 with quinons as electron acceptor or 1.1.3 with oxygen as acceptor particularly preferred:
cellobiose: quinone-l-oxidoreductase 1.1.5.1.
Furthermore usable are enzymes of the class 1.2, particularly preferred 1.2.3 with oxygen as acceptor or 1.3, particularly preferred 1.3.3 with oxygen as acceptor and 1.3.5 with quinone as acceptor, particularly preferred: bilirubin oxidase 1.3.3.5,
Furthermore usable are enzymes of the class 1.9 particularly preferred: cytochrome-oxidase 1.9.3.
Furthermore preferred are enzymes of the class 1.1.2 and oxygenases, lipoxygenases 1.13, 1.14 and 1.1.5, which use superoxid as electron acceptor, particularly preferred:
superoxide dismutase 1.15.11 and 1.16 particularly preferred: ferrioxidase e.g. ceruloplasmin 1.16.3.1.
Especially preferred being the enzymes of sub-class 1.10. such as catechol oxidase (tyrosinase) (1.10.3.1), L-ascorbate oxidase (1.10.3.3), O-aminophenol oxidase (1.10.3.4) and laccase (benzenediol: oxygen oxidoreductase) (1.10.3.2).
Other particularly preferred enzymes are those of group 1.11.
Especially preferred here are cytochrome C peroxidases (1.11.1.5), catalase (1.11.1.6), peroxidase (1.11.1.7), iodide peroxidase, (1.11.1.8), glutathione peroxidase (1.11.1.9), chloride peroxidase (1.11.1.10), L-ascorbate peroxidase (1.11.1.11), phospholipid hydroperoxide glutathione peroxidase (1.11.1.12), manganese peroxidase (1.11.1.13) and diarylpropane peroxidase (ligninase, lignin peroxidase) (1.11.1.14).
According to the invention the term enzyme comprises also enzymatic active proteins or peptides or prosthetic groups of enzymes. The enzymes used can stem from wild type microorganism strains or genetically manipulated strains.
The mentioned enzymes are commercial available or can be produced according to state-of-the-art production processes.
As production organisms for producing these enzymes plants, bacteria and fingi, parts of unicellular or multi-cellular organisms or or cell cultures can be used.
Particularly prefrred production organisms manly for the production of lignolytic enzymes such as laccases, lignin peroxidases and manganese peroxidases etc. are, for example, white rotting fungi like e.g. Pleurotus, Phlebia, Trametes, Agaricus, Lentinus, Botrytis, Cryphonectria, Hypholoma, Heterobasidion, Phanerochaete.
As co-substrates air, oxygen, ozone, peroxides, such as H₂O_2,organic peroxides, peracids such as peracetic, performic, persulfuric, pernitric, metachloroperoxybenzoic and perchloric acid, per compounds such as perborates, percarbonates or persulfates, or oxygen species and the radicals thereof such as the OH, OOH⁻ and OH⁺ radical, superoxide (O₂—), dioxygenyl cation (O₂ ⁺), singlet oxygen can be used.
As carbonyl compounds (ketones, aldehydes)—with exeption of benzophenones and benziles—are particularly preferred compounds of the general formula XIV:
The R¹and R²groups can be equal or different and denote aliphatic or aromatic groups. Moreover, the R¹and R²groups can form a ring containing besides carbon also heteroatoms such as nitrogen, oxygen and sulfur.
Particularly preferred are 1,2-diketones (formula XV), 1,3-diketones (formula XVI), polyketones (polyketides) and the tautomeric enols (formula XVII):

wherein the R³to R⁶groups, once again, can be equal or different and denote aliphatic or aromatic groups. Moreover, groups R³and R⁴and groups R⁵and R⁶, together, can form a ring containing besides carbon also heteroatoms such as nitrogen, oxygen or sulfur. The possibility of tautomerization or formation of a resonance hybrid is particularly important in this case. Besides general carbonyl compounds, particularly preferred are ketones, such as, in general hydroxyketones, α,β-unsaturated ketones, oxycarboxylic acids, quinones and halogenated ketones.
Particularly preferred among these are the following:
Acetone, methyl ethyl ketone, diethyl ketone, methyl n-butyl ketone, methyl isobutyl ketone, cyclohexanone, cyclopentanone, 2-methylcyclohexanone, 3-methylcyclohexanone, 4-methylcyclohexanone, dihydroxyacetone, diacetyl monohydrazone, diacetyl dihydrazone, acetophenone, p-hydroxyacetophenone, 1-phenyl-3-butanone, 3-pentanone, 4-heptanone, 2-nonanone, cycloheptanone, cyclooctanone, cyclodecanone, cyclododecanone, dimethyl ketone, ethyl propyl ketone, methyl amyl ketone, acetylacetone, pinacoline, methyl isopropyl ketone, methyl isoamyl ketone, ethyl amyl ketone diisopropyl ketone, diisobutyl ketone, methyl vinyl ketone, methyl isopropenyl ketone, mesityl oxide, isophorone, hydroxyacetone, methoxyacetone, 2,3-pentanedione, 2,3-hexanedione, phenylacetone, propiophenone, benzophenone, benzoin, benzil, 4,4′-dimethoxybenzil, 4′-methoxyacetophenone, 3′- methoxyacetophenone, O-ethylbenzoine, (2-methoxyphenyl)acetone, (4-methoxyphenyl)acetone, methoxy-2-propanone, glyoxylic acid, benzyl glyoxylate, benzylacetone, methyl benzyl ketone, methylcyclohexyl ketone, 2-decanone, dicyclohexyl ketone, 3,3-dimethyl-2-butanone, methyl isobutyl ketone, methyl isopropyl ketone,2-methyl-3-heptanone, 5-methyl-3-heptanone, 6-methyl-5-hepten-2-one, 5-methyl-2-hexanone, 3-nonanone, 5-nonanone, 2-octanone, 3-octanone, 2-undecanone, 1,3- dichloroacetone, 1-hydroxy-2-butanone, 3-hydroxy-2-butanone, 4-hydroxy-4-methyl-2-pentanone, 2-(1S)-adamanantone, anthrone, bicyclo(3.2.0)hept-2-en-6-one, cis-bieyclo(3.3.0)octan-3,7-dione, (1S)- (−)-camphor, p-chloranil, cyclobutanone, 1,3-cyclohexanedione, 1,4-cyclohexanedione monoethylene ketal, dibenzosuberone, ethyl 4-oxocyclohexanecarboxylate, 9-fluorenone, 1,3-indandione, methyl- cyclohexanone, phenylcyclohexanone, 4-propylcyclohexanone, 1,2,3,4-tetrahydro-1-naphthalenone, 1,2,3,4-tetrahydro-2-naphthalenone, 3,3 ,5-trimethylcyclo-hexanone, 3-acetoxy-2-cyclohexen-1-one, benzylideneacetone, (R)-(−)-carvone, (S)-(−)carvone, curcumin, 2-cyclohexen-1-one, 2,3-diphenyl-2- cyclopropen-l-one, 2-hydroxy-3-methyl-2-cyclopentene-1-one, isophorone, α-ionone, β-ionone, 3-methoxy-2-cyclohexen-1-one, 3-methyl-2-cyclopenten-1-one, 3-methyl-3-penten-2-one, (R)-(+)-pulegone, tetraphenyl-2,4-cyclopentadien-1-one, 2,6,6-trimethyl-2-cyclohexen-1,4-dione, 2-acetylbenzoic acid, 1-acetylnaphthalene, 2-acetylnaphthalene, 3′-aminoacetophenone, 4′-aminoacetophenone, 4′-cyclohexylacetophenone, 3′, 4′-diacetoxyacetophenone, diacetylbenzene, 2′,4′-dihydroxyacetophenone, 2′,5′-dihydroxyacetophenone, 2′,6′-dihydroxyacetophenone, 3,4-dimethoxyacetophenone, 2′-hydroxyacetophenone, 4′-hydroxyacetophenone, 3′-methoxyacetophenone, 4′-methoxyacetophenone, 2′-methylacetophenone, 4′-methylacetophenone, 2′-nitroacetophenone, 3′-nitroacetophenone, 4′-phenylacetophenone, 3′,4′,5′-trimethoxy-acetophenone, 4′-aminopropiophenone, benzoylacetone, benzoylpropionic acid, benzylideneacetophenone, cyclohexyl phenyl ketone, desoxybenzoin, 4′,4′-dimethoxybenzil, 1,3-diphenyl-1,3-propanedione, ethylbenzoyl acetate, ethyl phenylglyoxylate, 4′-hydroxypropiophenone, 1,3-indandione, 1-indanone, isopropyl phenyl ketone, 6-methoxy-1,2,3,4- tetrahydronaphthalen-1-one, methylphenyl glyoxylate, phenylglyoxylonitrile, 1-phenyl-1, 2-propanedione 2-oxime, valerophenone, 2-acetyl-γ-butyrolactone, 2-acetylpyrrole, 1-benzylpiperidin-4-one, dehydroacetic acid, 3,4-dihydro-4,4-dimethyl-2H-pyran-2-one, 1,4-dihydro-4-pyridinone, N-eth-oxycarbonyl-4-piperidinone, 2-methyl furyl ketone, 5-hydroxy-2-hydroxymethyl-4H-pyran-4-one, 3-hydroxy-2-methyl-4-pyranone, 3-indolyl methyl ketone, isatin, 1-methyl-4-piperidinone, methyl 2-pyridyl ketone, methyl 3-pyridyl ketone, methyl 4-pyridyl ketone, methyl 2-thienyl ketone, phenyl 2-pyridyl ketone, phenyl 4-pyridyl ketone, tetrahydrofuran-2,4-dione, tetrahydro-4H-pyran-4-one, 2,2,6,6-tetramethyl-4-piperidone, xanthone, acenaphthene quinone, pyruvic acid, (1R)-(−)-camphor quinone, (1S)-(+)-camphor quinone, 3,5-ditert.butyl-o-benzoquinone, 1,2-dihydroxy-3,4- cyclobutendione, ethyl (2-amino-4-thiazolyl)glyoxylate, ethyl pyruvate, 2,3-hexanedione, 3,4- hexanedione, 3-methyl-2-oxobutyric acid, 3-methyl-2-oxovaleric acid, 4-methyl-2-oxovaleric acid, 2- oxobutyric acid, 2,3-pentandione, 9,10-phenanthrene quinone, acetoacetanilide,2-acetyl-y-butyrolactone, 2-acetylcyclo-pentanone, allyl acetoacetate, benzoylacetone, tert.butyl acetoacetate, 1,3-cyclopentanedione, diethyl 3-oxoglutarate, dimethyl acetylsuccinate, dimethyl 3-oxoglutarate, 1,3-diphenyl-1,3-propanedione, ethyl acetoacetate, ethyl benzoylacetate, ethyl butyrylacetate, ethyl 2-oxocyclohexanecarboxylate, ethyl 2-phenylacetoacetate, methyl acetoacetate, 2-methyl-1,3- cyclohexanedione, 2-methyl-1,3-cyclopentanedione, methyl isobutyrylacetate, methyl 3-oxopentanoate, methyl pivaloylacetate, 3-oxoglutaric acid, tetrahydrofuran-2,4-dione, 2,2,6,6-tetramethyl-3,5-heptanedione, 3-benzoylpropionic acid, 1,4-cyclohexanedione, dimethyl acetylsuccinate, ethyl levulinate, 2-aminoanthraquinone, anthraquinone, p-benzoquinone, 1,4-dihydroxyanthraquinone, 1,8-dihydroxyanthraquinone, 2-ethylanthraquinone, methyl-p-benzoquinone, 1,4-naphthoquinone, tetramethyl-p-benzoquinone, 2,2-dimethyl-1,3-dioxan-4,6-dione, 2-benzoylbenzoic acid, 3-benzoyl-propionic acid, 5,6-dimethoxyphthataldehydic acid, levulinic acid, methyl trans-4-oxo-2-pentenoate, phthalaldehydic acid, terephthalaidehydic acid, dibutyl maleate, dibutyl succinate, dibutyl phthalate, dicyclohexyl phthalate, diethyl acetamidomalonate, diethyl adipate, diethyl benzylmalonate, diethyl butylmalonate, diethylethoxymethylene-malonate, diethyl ethylmalonate, diethyl fumarate, diethyl glutarate, diethyl isopropylidenemalonate, diethyl maleate, diethyl malonate, diethyl methylmalonate, diethyl oxalate, diethyl 3-oxoglutarate, diethyl phenylmatonate, diethyl phthalate, diethyl pimelate, diethyl sebacate, diethyl suberate, diethyl succinate, diisobutyl phthalate, dimethyl acetylene-dicarboxylate, dimethyl acetylsuccinate, dimethyl adipate, dimethyl 2-aminoterephthalate, dimethyl fumarate, dimethyl glutaconate, dimethyl glutarate, dimethyl isophthalate, dimethyl malonate, dimethylmethoxy-malonate, dimethyl methylenesuccinate, dimethyl oxalate, dimethyl 3-oxoglutarate, dimethyl phthalate, dimethyl succinate, dimethyl terephthalate, ethylene glycol diacetate, ethylene glycol dimethacrylate, monoethyl fumarate, monomethyl malonate, monoethyl adipate, monomethyl phthalate, monomethyl pimelate, monomethyl terephthalate, 1,2-propylene glycol diacetate, triethyl methanetricarboxylate, trimethyl 1,2,3-propanetricarboxylate, 3-acetoxy-2-cyclohexen-1-one, allyl acetoacetate, allyl cyanoacetate, benzyl acetoacetate, tert.butyl acetoacetate, butyl cyanoacetate, chlorogenic acid hemihydrate, coumarin-3-carboxylic acid, diethyl ethoxy-carbonylmethanephosphonate, dodecyl gallate, dodecyl 3,4,5-trihydroxybenzoate, (2,3-epoxypropyl) methacrylate, (2-ethoxyethyl) acetate, ethyl acetamidocyanoacetate, ethyl 2-aminobenzoate, ethyl 3-aminopyrazol-4-carboxylate, ethyl benzoxylacetate, ethyl butyrylacetate, ethyl cyanoacetate, ethyl 2-cyano-3-ethoxyacrylate, ethyl cyanoformate, ethyl 2-cyanopropionate, ethyl 3,3-diethoxypropionate, ethyl 1,3-dithian-2-carboxylate, ethyl 2-ethoxyacetate, ethyl 2-furancarboxylate, ethyl levulinate, ethyl mandelate, ethyl gallate, ethyl 2-methyllactate, ethyl 4-nitrocinnamate, ethyl oxamate, ethyl 2-oxocyclohexanecarboxylate, ethyl 4-oxocyclohexane- carboxylate, ethyl 5-oxohexanoate, ethyl 2-phenylacetoacetate, ethyl 4-piperidinecarboxylate, ethyl 2-pyridinecarboxylate, ethyl 3-pyridinecarboxylate, ethyl 4-pyridinecarboxylate, ethyl thioglycolate, ethyl 3,4,5-trihydroxybenzoate, 2-hydroxyethyl methacrylate, 2-hydroxypropyl methacrylate, 3-indole acetate, 2-methoxyethyl acetate, 1-methoxy-2-propyl acetate, methyl 2-aminobenzoate, methyl 3-aminocrotonate, methyl cyanoacetate, methyl 4-cyanobenzoate, methyl 4-formylbenzoate, methyl 2-furancarboxylate, methyl isobutyrylacetate, methyl methoxyacetate, methyl 2-methoxybenzoate, methyl 3-oxopentanoate, methyl phenylglyoxylate, methyl phenyl-sulfinylacetate, methyl pivatoylacetate, methyl 3-pyridinecarboxylate, 5-nitrofurfurylidene diacetate, propyl gallate, propyl 3,4,5-trihydroxybenzoate, methyl 3-methylthiopropionate, acetamide, acetani-lide, benzamide, benzanilide, N,N-diethylacetamide, N,N-dimethylformamide, N,N-diethyl-3-methyl-benzamide, diethyltoluamide, N,N-dimethylacetamide, N,N-diphenylacetamide, N-methylformamide, N-methylformanilide, N-acetylthiourea, adipic acid diamide, 2-aminobenzamide, 4-aminobenzamide, succinic acid diamide, malonic acid diamide, N,N′-methylene diacrylamide, oxalic acid diamide, pyrazine-2-carboxamide, pyridine-4-carboxamide, N,N,N′,N′-tetramethylsuccinic acid diamide, N,N,N′,N′-tetramethylglutaric acid diamide, acetoacetanilide, benzohydroxamic acid, cyanoacetamide, 2-ethoxybenzamide, diethyl acetamidomalonate, ethyl acetamidocyanoacetate, ethyl oxamate, hippuric acid Na salt, N-(hydroxy-methyl)acrylamide, L-(−)-lactarnide, 2′-nitroacetanilide, 3′- nitroacetanilide, 4′-nitroacetanilide, paracetamol, piperine, salicylanilide, 2-acetyl-γ-butyrolactone, γ-butyrolactone, ε-caprolactone, dihydrocoumarin, 4-hydroxycoumarin, 2-(5H)-furanone, 2,5-dihydro-5-methoxy-2-furanone, phthalide, tetrahydrofuran-2,4-dione, 2,2,6-trimethyl-1,3-dioxin-4-one, γ-valerolactone, 4-amino-1,3-dimethyluracil, barbituric acid, O-benzyloxycarbonyl-N-hydroxysuccinimide, succinimide, 3,6-dimethyl-piperazin-2,5-dione, 5,5-diphenylhydantoin, ethyl 1,3-dioxoisoindoline-2-carboxylate, 9-fluorenylmethylsuccinimidyl carbonate, hydantoin, maleimide, 3-methyl-1-phenyl-2-pyrazolin-5-one, 1-methyl-2-pyrrolidone, methyluracil, 6-methyluracil, oxindole, phenytoin, 1-(2H)-phthalazinone, phthalimide, 2,5-piperazinedione, 2-piperidinone, 2-pyrrolidone, rhodanine, saccharin, 1,2,3,6-tetrahydrophthalimide, 1,2,3,4-tetrahydro-6,7-dimethoxyquinazolin-2,4-dione, 1,5,5-trimethyl-hydantoin, 1-vinyl-2-pyrrolidone, ditert.butyl dicarbonate, diethyl carbonate, dimethyl carbonate, dimethyl dicarbonate, diphenyl carbonate, 4,5-diphenyl-1,3-dioxol-2-one, 4,6- diphenylthieno-(3,4-d)-1,3-dioxo[-2-one 5,5-dioxide, ethylene carbonate, magnesium methoxide methyl carbonate, monomethyl carbonate Na salt, propenyl carbonate, N-allylurea, azodicarbonamide, N-benzylurea, biuret, 1,1′-carbonyldiimidazol, N,N-dimethylurea, N-ethylurea, N-formylurea, urea, N-methylurea, N-phenylurea, 4-phenylsemicarbazide, tetramethylurea, semicarbazide hydrochloride, diethyl azodicarboxylate, methyl carbamate, 1-(4-methoxyphenyl)-2-(2-methoxyphenoxy)ethanone and 1-(4-methoxyphenyl)-2-(2-methoxyphenoxy)ethanol.
Also preferred are anhydrides, such as the following:
Benzoic anhydride, benzene-1,2,4,5-tetracarboxylic acid-1,2,4,5-dianhydride, 3,3′,4,4′-benzophenonetetracarboxylic anhydride, succinic anhydride, butyric anhydride, crotonic anhydride, cis-1,2-cyclo-hexanedicarboxylic anhydride, ditert.butyl dicarbonate, dimethyl dicarbonate, dodecenylsuccinic anhydride, Epicon B 4400, acetic anhydride, glutaric anhydride, hexanoic anhydride, isatoic anhydride, isobutyric anhydride, isovaleric anhydride, maleic anhydride, 1,8-naphthalenedicarboxylic anhydride, 3-nitrophthalic anhydride, 5-norbomene-2,3-dicarboxylic anhydride, phthalic anhydride, 2-phenylbutyric anhydride, pivalic anhydride, propionic anhydride, cis-1,2,3,6-tetrahydrophthalic anhydride and valeric anhydride.
Application Fields of the Oxidation and/or Bleaching System According to the Invention are Above All:
I) the use in the bleaching of cellulose/wood pulp,
II) the use in the treatment of different wastewaters (pulp and paper industry, other),
III) the use in the preparation of lignin solutions or gels, of the corresponding binders/adhesives and of wood-based composites,
IV) the use as enzymatic deinking system,
V) the use as oxidation systems in organic synthesis,
VI) the use as bleaching agent in detergents,
VII) the use as bleaching and/or oxidation system in textile industry, inclusive stone washing and bleaching of fabrics and the general oxidative treatment of wool (e.g. bleaching),
VIII) the use in coal liquefaction,
due to the fact, that the new oxidation and/or bleaching system according to the invention doesn't exhibit the disadvantages of pure chemical systems (e.g. environmental safety problems) or other enzyme-based systems ( e.g. low performance, high costs).
It was surprisingly found that by the use of the new oxidation and/or bleaching system according to the invention comprising of
oxidoreductases such as laccases and/or peroxidases—seperately or in combination—in the presence of their respective co-substrates like O_2,air, H₂O₂, organic peroxides, peracids etc. and comprising of
enhancer compounds from the class of oxocarbons, from the class of urazoles and hydrazides, from the class of hydantoins and the class of nitril (Cyan)-compounds, and comprising additionally of
carbonyl compounds such as ketones, aldehyds,
whereby the combination of enzyme, co-substrate, enhancer compound and carbonyl compound generate active oxygen species like dioxiranes, dioxetanes, peroxy-compounds etc. or form (according e.g. to the literature: K. Deuchert et. al., Angewandte Chemie 90, S. 927-938, 1978) also other reactive compounds or transition states like radicals (kation radicals, anion radicals) or reactive (red/ox-active) neutreal compounds as oxidizing and/or bleaching agents
a very significant delignification of pulp, a bleaching of high yield pulps, a bleaching of recycled wood fibres afterdeinking processes an oxidative polymerisation of lignin or lignin-derived substances including decolorisation and or detoxification during treatment of chemical pulp or high yield pulp waste waters or a decolorisation and or detoxification of other waste waters, an oxidative polymerisation of the available polyphenyl-propan substances during the production of wood-based composites etc. a release of the dyed particles from fibres during a deinking process and a liquefaction of coal could be obtained.
Furthermore it was surprisingly found that the new oxidation and/or bleaching system according to the invention shows a significant and selective oxidation efficiency during its use as oxidizing agent in organic synthesis , shows a high bleaching efficency as bleaching additive in detergents and shows a high oxidation power (bleaching efficiency) during oxidative treatment of textiles including wool.
The method described in DE 19820947.9-45 can be distinguished from the present invention through the addition of suitable ketone (generally aliphatic) coumpounds which are not used there. No benzophenones, benzils and some other organic carbonyl compounds which were described in the older application are used due to the fact that they show no effect using the new system.
A further advantage of the new system is that no oxygen pressure has to be applied.
For the first time a catalytic behaviour at high delignification performance ( e.g. 30% kappa reduction at 200 g per ton oxocarbon dosage) of the new system according to the invention could also be shown, provided the addition of suitable ketone compounds.,
Furthermore the addition of primary, secondary or tertiary amines can be of some advantage (see literature: K. Deuchert et. al., Angewandte Chemie 90, S. 927-938, 1978.
As combining systems in combination with the new oxidation and/or bleaching system according to the invention the enzymatic system HOS (hydrolase mediated oxidation system), described in W098/59108, consisting of lipases or other hydrolases, peroxide, ketones and fatty acids/fats, or enzyme-based systems, described in DE 101 26 988.9 ca be used.

DESCRIPTION OF VARIOUS APPLICATIONS OF THE ENZYME COMPONENT SYSTEM OF THE INVENTION

I) the use in the bleaching of cellulose/wood pulp,
II) the use in the treatment of different wastewaters (pulp and paper industry, other),
IV) the use in the preparation of lignin solutions or gels, of the corresponding binders/adhesives and of wood-based composites,
IV) the use as enzymatic deinking system,
V) the use as oxidation systems in organic synthesis,
VI) the use as bleaching agent in detergents,
VII) the use as bleaching and/or oxidation system in textile industry, inclusive stone washing and bleaching of fabrics and the general oxidative treatment of wool (e.g. bleaching),
VIII) the use in coal liquefaction,
I) Use of the Enzymatic Oxidation and/or Bleaching System According to the Invention in Pulp Bleaching
Wood pulp is currently produced mainly by the sulfate and sulfite processes. By both processes, pulp is made by cooking at high temperature and under pressure. The sulfate process involves the addition of NaOH and Na₂S, whereas the sulfite process uses Ca(HSO₃)₂, +SO₂, although the sodium and ammonium hydrogen sulfite salts are currently used because of their higher solubility.
The main objective of all processes is the removal of lignin from the plant material, wood or annual plants used.
The lignin, which together with the cellulose and hemicellulose forms the main constituent of the plant material (stalks and stems), must be removed, because it is otherwise not possible to produce nonyellowing, mechanically highly resistant papers.
The processes for making high yield wood pulp involve the use of stone grinders (groundwood) or of refiners (TMP=thermomechanical pulp) which after an appropriate pretreatment (chemical, thermal or thermechemical) defibrillate the wood by milling.
These wood pulps still contain most of the lignin. They are used primarily for the production of newspapers, magazines etc.
The possibilities of using enzymes for lignin degradation have been under investigation for several years. The mechanism of action of such lignolytic systems was discovered only a few years ago, when it became possible to obtain sufficient amounts of enzymes from the white rotting fungus Phanerochaete chryosporium by use of proper culturing
conditions and the addition of inductors. This is how the hitherto unknown lignin peroxidases and manganese peroxidases were detected. Because Phanerochaete chryosporium is a very effective lignin degrader, attempts have been made to isolate its enzymes and use them in purified form for lignin degradation. This was unsuccessful, however, because it was found that the enzymes primarily cause repolymerization of lignin and not its degradation.
The same is true for other lignolytic enzyme species, such as the laccases which degrade lignin oxidatively with the aid of oxygen rather than hydrogen peroxide. It was found that similar processes are at work in all cases, namely that radicals are formed which then react with each other causing the mentioned polymerization.
Currently, there are only processes based on the use of in-vivo systems (fungal systems). Optimization attempts were directed mainly toward biopulping and biobleaching.
By biopulping is meant the treatment of wood chips with live fungal systems. There are two kinds of application forms:
1 . Pretreating the wood chips before charging them to the ref iners or milling for the purpose of saving energy in high yield wood pulp production (for example, TMP or groundwood). Another advantage is the usually achieved improvement of mechanical properties of the stock, and a drawback is that the final brightness is worse.
2. Pretreating the wood chips (softwood/hardwood) before wood pulp cooking (kraft process, sulfite process). Here, the objective is to reduce the amount of digestion chemicals, to improve digestion capacity and extended cooking. The advantages include improved kappa number reduction following digestion compared to digestion without pretreatment.
The drawbacks of these processes are clearly their long treatment times (several weeks) and particularly the unsolved problem of risk of contamination during the treatment, if it is desired to omit the uneconomical sterilization of the wood chips.
Biobleaching also uses in-vivo systems. Before bleaching, the digested pulp (softwood/hardwood) is inoculated with the fungus and treated for a period of days or weeks. Only after such a long treatment time is it possible to observe a drop in kappa number and a significant improvement in brightness, so that the process is uneconomical for implementation in current bleaching sequences.
Another application, mostly carried out with immobilized fungal systems, is the treatment of pulp production wastewaters, particularly bleaching plant wastewaters, for the purpose of decolorizing them and reducing the AOX value (reducing the amount of chlorinated compounds in the wastewater, compounds which were used for chlorine or chlorine dioxide bleaching). It is also known to use hemicellulases and particularly xylanases and mannases as bleach boosters.
These enzymes act mainly on the reprecipatated xylan, which after the cooking process partly covers the residual lignin, for the purpose of degrading it and thus improving accessibility to the lignin of the bleaching chemicals (primarily chlorine dioxide) used in the subsequent bleaching sequences. The savings in bleaching chemicals demonstrated in the laboratory have been confirmed on a large scale only to a limited extent so that this type of enzyme must also be classified as a bleaching additive.
Patent application PCT/EP 87/00635 describes a system for removing lignin from lignin-cellulose-containing material with simultaneous bleaching. The system is based on the use of lignolytic enzymes from white rotting fugi with the addition of reducing agents, oxidants and phenolic compounds as mediators.
According to DE 4 008 893 C2, in addition to the redox system, mimicking substances are added which simulate the active center (prosthetic group) of lignolytic enzymes. In this manner, a marked improvement in performance is achieved.
According to patent application PCT/EP 92/01086, additional improvement is achieved by use of a redox cascade with the aid of phenolic or nonphenolic aromatics “balanced” in terms of their oxidation potential.
All three processes are limited in regard to their applicability on an industrial scale in that they must be used at low wood pulp consistency (up to a maximum of 4%) and, in the case of the last two applications also by the risk of leaching out metals when chelating agents are used, the metals possibly causing peroxide decomposition in the subsequent peroxide bleaching stages.
WO 94/12619, WO 94/12620 and WO 94/12621 disclose processes in which the peroxidase activity is increased with enhancers. Enhancers are characterized in WO 94/12619 in terms of their half-life.
According to WO 94/12620, enhancers are characterized by the formula A=N—N═B where N means nitrogene A and B are defined cyclic groups. According to WO 94/12620, enhancers are organic chemicals containing at least two aromatic rings of which at least one is substituted with defined groups.
All three patent applications concern dye transfer inhibition and the use of enhancers together with peroxidases as detergent additives or detergent compositions used in the detergent sector.
Although the applicability to lignin is mentioned in the specification of the applications, our own tests with the substances actually disclosed in these applications have shown that the claimed mediators are ineffective in increasing the bleaching action of peroxidases in the treatment of lignin-containing materials!.
WO 94/29510 and WO 96/18770 describe a process for enzymatic delignification whereby the enzymes are used together with mediators. The mediators disclosed are, in general, compounds characterized by the structure NO—, NOH— or NRNOH.
Among the mediators disclosed in WO 94/29510 and WO 96/18770, 1-hydroxy-1H-benzotriazole (HOBT) gave the best delignification results. HOBT, however, has several drawbacks:

- it is available only at a relative high price and in insufficient quantities,
- under deliginification conditions, it reacts forming 1H-benzotriazole and other colored products, this compound shows relatively low degradability and could, in large amounts, present a pollution problem,
- to a certain degree, it harms the enzymes,
- its delignification velocity is not very high.

Other mediator of the described NO—, NOH— and HRN—OH type do not show most of these drawbacks, but still have the disadvantage that a relatively large amount of chemicals must be used and, particularly, that because of their physiological reactivity they may not be entirely harmless (mostly because of NO— radical formation).
It is therefore desirable to provide systems for modifying, degrading or bleaching lignin, lignin-containing materials or similar substances, which do not have the said drawbacks or present them only to a minor degree.
This can be reached using the new oxidation and/or bleaching system according to the invention which doesn't show the mentioned disadvantages, i.e. it could be shown quite surprisingly, that using the enzyme-based system according to the invention at suitable combination of the components better delignification and bleaching results are achieved compared to the abovesaid other enzymatic systems, and the said drawbacks are negligible.
IIa) Use of the Enzymatic Oxidation and/or Bleaching System According to the Invention in the Enzymatic Treatment of Special Waste Waters (Paper Industry Waste Waters from e.g. Groundwood Plants or Refiner Plants)
IIb) Use in the Enzymatic Treatment of Other Industrial Waste Waters
Unlike most enzymes, oxidases and peroxidases exhibit low substrate specificity, namely they can convert a wide range of substances, usually of phenolic nature. Without mediators, oxidases as well as many peroxidases show the tendency to polymerize phenolic substances via free radical-induced polymerization, a property which is attributed, for example, to laccase, belonging to the group of oxidases, also in nature. The ability to polymerize appropriate substances, for example lignins, namely to increase the size of the molecules involved by “coupling reactions”, can be utilized, for example, for the treatment of lignin-containing wastewaters in the paper industry such as TMP wastewater (wastewater from the production of thermomechanical pulp by means of refiners) and of grinder wastewater from mechanical wood pulping units.
The water-soluble lignin compounds (polyphenolpropanes) contained in these wastewaters are mainly responsible for the high COD (chemical oxygen demand) and cannot be removed by conventional technology. In the water treatment plant and in the downstream waters, they are not degraded at all or they are degraded only very slowly. At very high concentrations, these compounds can even inhibit the bacteria in a water treatment plant and thus create problems.
In this application, the enzyme action can be observed immediately by a rapid development of turbidity in the wastewater being treated, caused by an enlargement and thus insolubilization of lignin molecules. The target molecules (polymerized lignin) thus enlarged in molecular weight by enzymatic catalysis can be removed by appropriate treatments (by flocculation, by precipitation with, for example, aluminum sulfate/sodium aluminate, optionally in the presence of cationic or anionic polyelectrolytes or by sedimentation). The wastewater then shows a markedly reduced COD. Upon disposal, such wastewater causes less pollution, namely it increases the certainty of remaining below the permissible COD limits. Thus is particularly important for not infrequently used “procedures” run at the limit.
For this treatment, for example with laccase, the cost of removing the reaction products of the enzymatic treatment by flocculation, sedimentation or precipitation or a combination of several such methods constitutes by far the predominant part of the overall cost of the process.
All other industrial wastewaters containing phenolic or, in general, oxidizable substances (for example, lignin, dyes etc) can, in principle, be treated with, for example, the abovesaid oxidoreductases. Such treatment can be applied to wastewaters from grape presses, olive presses, dyeing plants in the textile industry, wastewaters from pulping plants etc. If at all possible, to attain maximum efficiency, polluted streams should be treated before they are combined with other wastewaters.
To this system are added other special compounds ( polymerization catalysts) which serve as condensation nuclei and can substantially enhance lignin polymerization so that the main objective of this enzymatic wastewater treatment, which is to use the lowest possible amount of the cost-intensive precipitant, can be attained.
Such compounds are normally phenols, phenol derivatives and polycyclic phenols which have more than one oxidizable phenolic group.
Such polymerisation catalysts, which are claimed here, are also described in WO98/59108 and DE 10126988.9.
Also in this case we have found, quite surprisingly, that when the new enzyme-based oxidation and/or bleaching system according to the invention is used by employing a special
Also in this case we have found, quite surprisingly, that when the new enzyme-based oxidation and/or bleaching system according to the invention is used by employing a special combination of the components, much higher efficiency in treatment of the above mentioned waste waters than be attained than with the above-described enzymatic systems.
III) Use of the Enzymatic Oxidation and/or Bleaching System According to the Invention in the Preparation of Lignin Solutions or Gels, of the Corresponding Binders/Adhesives and of Wood-Based Composites
The object of the present invention is to provide a process for enzymatic polymerization and/or modification of lignin or lignin-containing materials, for example for use in the production of wood compositions or wood-based composites such as, for example, fiber board from disintegrated wood or particle board from wood chips or wood pieces (chipboard, plywood, wood composite beams).
It is known from the literature and patents, for example WO 94/01488, WO 93/23477, WO 93/25622 and DE 3 037 992 C2 that laccases, lignin peroxidases or peroxidases can be used for this purpose. The main drawbacks, particularly in the case of laccases and lignin peroxidases, are the difficulty of preparing these enzymes and the low yields even of genetically modified systems.
We have now found, quite surprisingly, that. here, too, the enzymatic oxidation and or bleaching syste of the invention shows much better performance compared to the prior-art enzymatic systems for the polymerization and/or modification of lignin and/or lignin-containing materials.
To this end, the enzyme component system of the invention is brought together with lignin (for example, with lignosulfates and/or unevaporated or evaporated sulfite waste liquor and/or sulfate lignin→kraft lignin, for example induline) and/or with lignin-containing material. The lignin and/or the lignin-containing material can either be preincubated at an elevated pH, namely above pH 8 and preferably at a pH between 9.5 and 10.5, at 20 to 100° C. (preferably at 60 to 100° C.) after which the pH is reduced to below pH 7, depending on the optimum pH range for enzyme activity of the enzyme-based system or, if the activity optimum of the enzyme-based system is on the alkaline side, the system and the lignin and/or the lignin-containing material are brought together immediately, without pretreatment. The purpose of the pretreatment or treatment under alkaline pH conditions is to utilize the substantially easier solubilization of lignin at these higher pH values. This is a major advantage for the use according to the invention, because it is thus possible to work without an organic solvent.
In other words, the main purpose of the described bringing together of the enzyme component system and the lignin and/or lignin-containing material is to achieve activation of the substrates (polyphenylpropanes) by oxidation, namely to convert the lignin and/or the lignin-containing material by free radical-induced polymerization (modification) into an activated and active binder which then when brought together with the wood fibers and/or wood parts to be bonded (cemented together) can be cured under the action of pressure and elevated temperature to give solid wood-based composites such as the abovesaid wood products, for example fiber boards and particle boards. The main advantage consists of reducing, or producing savings in, the amount of urea-formaldehyde resins normally used, for example, for gluing in the production of chipboard, which resins, besides being toxic, have only limited moisture resistance, or of phenol-formaldehyde resins which exhibit unfavorabie swelling properties and require long pressing times (once again, besides the question of toxicity).
The polymerizing and/or modifying action of the enzyme component system can be additionally enhanced by addition of certain chemical polymerization catalysts, for example polydiphenylmethane diisocyanate (PMDI) and other polymerization catalysts used also for the polymerization of lignin in lignin-containing wastewaters. Such substances consist of phenols, phenol derivatives or other polycyclic phenolic compounds with a number of oxidizable hydroxyl groups, as already indicated hereinabove (wastewater treatment).
IV) Use of the Enzymatic Oxidation and/or Bleaching System According to the Invention as an Enzymatic Deinking System
In principle, by deinking, which is currently always run in a conventional manner by flotation, is meant a two-step process.
Its objective is to remove printing ink and other dye particles from the waste paper. The waste paper used in most cases is paper collected domestically and consists mainly of newspapers and magazines.
In the first treatment step, the dye particles adhering to the paper fibers are removed primarily by mechanical/chemical means. This is accomplished by “recycling” the paper as a uniform fibrous slurry, namely by disintegrating (comminuting) the waste paper in pulpers, drums or the like with simultaneous addition of chemicals capable of enhancing removal and preventing yellowing and thus also acting as bleaching chemicals, namely sodium hydroxide solution, fatty acid, water glass and hydrogen peroxide (H₂O₂). Here, the fatty acid acts as a fiber dye particle collector and in the second treatment step, the flotation, also as foaming agent.
After the waste paper has been disintegrated and the said chemicals have been allowed to act for a certain length of time, the flotation is carried out in special flotation vessels by injecting air. During this process, the dye particles adhere to the foam bubbles and are removed together with the bubbles. The dye is thus separated from the paper fibers. Currently, this operation is preferably carried out at a neutral pH, which makes it necessary to use certain detergents in place of the fatty acids.
It is known from the literature (WO 91/14820, WO 92/20857) to use an oxidoreductase or laccase system characterized primarily by the addition of special substances which cause the optimum pH for the action of laccase obtained from Trametes versicolor, which normally is in the range of about pH 4-5, to shift into the slightly alkaline range (pH 8 to 8.7). This, on the one hand, is an important prerequisite for use in the deinking system because of the CaSO₄problems arising below pH 7 and, on the other, does not optimize the action of laccase in the polymerizing or depolymerizing sense, but only produces a certain swelling of the fibers. Such swelling (which is one of the main actions of sodium hydroxide in purely chemical deinking systems) is a primary performance characteristic of the dye-removing mechanism.
The only other additives required for this enzymatie system employing oxidoreductases are the detergents needed to produce foam. Nearly all suitable detergents also exert a dye-removing action. Moreover, in conventional deinking systems the use of sodium hydroxide and peroxide improves brightness as a result of the bleaching action of these chemicals. This bleaching action cannot be achieved with the prior-art enzyme system because of the nature of the system.
We have now found, quite surprisingly, that by appropriate selection of the components the enzymatic oxidation and/or bleaching system of the invention exceeds the efficiency of other enzymatic deinking systems, particularly those with oxidoreductases and those applied to lignin-containing deinked pulp and at least in part compensates for the advantage of bleaching with purely chemical systems.
This means that it is possible to provide a system offering environmentally friendly deinking under neutral pH conditions and thus better post-bleaching, better pulp properties etc and good performance similar to that of purely chemical systems.
In this case, a further improvement of printing ink removal can be attained by the aforesaid addition of special substances mostly of a phenolic nature and, in particular, containing several hydroxyl groups, which are also used as polymerization catalysts in enzymatic wastewater treatment and general polymerization reactions, as in the production of binders/adhesives from lignin or lignin-containing substances primarily for the preparation of wood-based composites.
V) Use of the Enzymatic Oxidation and/or Bleaching System According to the Invention as an Oxidation System in Organic Synthesis
Recently, enzymes have increasingly been used for chemical reactions in organic synthesis. A few examples showing a variety of oxidative reactions that can be carried out with enzymatic systems can be found in: Preparative Biotransformations (Whole Cell and Isolated Enzymes in Organic Synthesis), S. M. Roberts, K. Wiggins and G. Casy, J. Wiley & Sons Ltd, 1992/93;
Organic Synthesis with Oxidative Systems, H. L. Holland, VCH, 1992; and
Biotransformations in Organic Chemistry, K. Faber, Springer Verlag [publisher], 1992:
1) Hydroxylation Reactions
a) Synthesis of alcohols
b) Hydroxylation of steroids
c) Hydroxylation of terpenes
d) Hydroxylation of benzenes
e) Hydroxylation of alkanes
f) Hydroxylation of aromatic compounds
g) Hydroxylation of double bonds
h) Hydroxylation of nonactivated methyl groups
i) Dihydroxylation of aromatie compounds
2) Oxidation of Unsaturated Aliphatics
a) Preparation of epoxides
b) Preparation of compounds by epoxidation
c) Preparation of arene oxides
d) Preparation of phenols
e) Preparation of cis-dihydrodiols
3) Baeyer-Villiger Oxidations
a) Baeyer-Villiger conversion of steroids
4) Oxidation of Heterocycles
a) Transformation of organic sulfides
a) Oxidation of sulfur compounds
b) Oxidation of nitrogen compounds (formation of N-oxides etc.)
d) Oxidation of other heteroatoms
5) Carbon-Carbon Dehydrogenation
a) Dehydrogenation of steroids
6) Other Oxidation Reactions
a) Oxidation of alcohols and aldehydes
b) Oxidation of aromatic methyl groups to aldehydes
c) Oxidative coupling of phenols
c) Oxidative degradation of alkyl chains (β-oxidation etc.)
e) Formation of peroxides or percompounds
f) Initiation of free-radical induced chain reactions.
Here, too, we found, quite surprisingly, that with the aid of the enzymatic oxidation and/or bleaching system of the invention it is possible to carry out many oxidation reactions exemplified hereinabove.
VI) Use of the Enzymatic Oxidation and/or Bleaching System According to the Invention as Bleaching Agent in Detergents
Conventional bleaching systems in domestic detergents are unsatisfactory, particularly in the low-temperature range. Below a washing temperature of 60° C., the standard bleaching agent, i.e., H₂O₂/sodium perborate/sodium percarbonate must be activated by the addition of chemical bleach activators, such as TAED and/or SNOBS. Also, the need exists for more highly biodegradable, bio-compatible bleaching systems and systems for low-temperature washing that can be used in small amounts. Whereas enzymes are already being used industrially for protein-starch-fat solution and for fiber treatment in the washing process, no enzymatic system is currently available for detergent bleaching. WO 1/05839 describes the use of different oxidative enzymes (oxidases and peroxidases) to prevent dye transfer. As is known, peroxidases are capable of “decolorizing” different pigments (3-hydroxyflavone and betaine are decolorized by horseradish peroxidase and carotene by peroxidase). Said patent application describes the decolorization (also referred to as bleaching) of textile dyes removed from the laundered goods and present in the washing liquor (conversion of a colored substrate into a noncolored, oxidized substance). In this case, the enzyme is said to have the advantage over, for example, hypochlorite which attacks dyes in or on the fabric, in that the enzyme decolorizes only the dissolved dyes. Hydrogen peroxide or an appropriate precursor generating hydrogen peroxide in situ participates in the catalysis of the decolorization. The enzyme reaction can be enhanced somewhat by addition of other oxidizable enzyme substrates, for example metal ions such as Mn⁺⁺, halide ions, such as Cl⁻ or Br⁻ or organic phenols, such as p-hydroxycinnamic acid and 3,4-dichlorophenol. In this case, it is postulated that short-lived radicals or other oxidized states of the added substrate are formed and are responsible for the bleaching or other modification of the colored substance.
U.S. Pat. No. 4,077,6768 describes the use of iron porphin, hemin chloride, iron phthalocyanines or derivatives thereof together with hydrogen peroxide for preventing dye transfer. These substances, however, are rapidly destroyed by excess peroxide, and for this reason hydrogen peroxide formation must occur in a controlled fashion.
Processes are known from WO 94/12619, WO 94/12620 and WO 94/12621 whereby the activity of the peroxidase is enhanced by means of enhancers. Such enhancers are characterized in WO 94/12620 in terms of their half-life. According to WO 94/12621, enhancers are characterized by the formula A=N—N═B where N means nitrogen and A and B are defined cyclic groups. According to WO 94/12620, enhancers are organic chemicals containing at least two aromatic rings of which at least one is substituted with defined groups. All three patent applications concern dye transfer inhibition and the use of enhancers together with peroxidases as detergent additives or detergent compositions used in the detergent sector. The combination of these enhancers is limited to peroxidases. The use of mixtures containing peroxidases is also known from WO 92/18687. A special system of oxidases and of appropriate substrates such as hydrogen peroxide is described in German Unexamined Patent Application DE 42 31 761. German Unexamined Patent Application DE 19 18 729 concerns another special detergent system consisting of glucose and glucose oxidase or of starch, aminoglucosidase and glucose oxidase (GOD) and of added hydroxylamine or a hydroxylamine compound, wherein the hydroxylamine or the derivatives thereof serve to inhibit the catalase that is often present in GOD. Hydroxylamine and the derivatives thereof have definitely not been described as mediator additives.
Finally, WO 94/29425, DE 4445088.5 and WO 97/48786 concern multicomponent bleaching systems for use with detergents and which consist of oxidation catalysts and oxidants and of aliphatic, cycloaliphatic, heterocyclic or aromatic compounds containing NO—, NOH— or H—NR—OH groups.
All hitherto known “enzymatically enhanced” detergent-bleaching systems have the drawback that their cleaning and bleaching action is still unsatisfactory and that the mediators must be used in excessive amounts which may cause environmental and economic problems.
We have now found, quite surprisingly, that the enzymatic oxidation and/or bleaching system of the invention exceeds the performance of the aforesaid oxidoreductase-mediator systems and does not have the said drawbacks of the prior art,
VII) Use of the Enzymatic Oxidation and/or Bleaching System According to the Invention in the Bleaching and/or Decolorization of Textile Fabrics
Enzymes are currently being used to an increasing extent in various applications in the textile industry, For example, the use of amylases in the desizing process is of great importance, because the use of strong acids, alkalies or oxidants is thereby avoided.
Similarly, cellulases are used for biopolishing and biostoning, a process which is mostly employed together with conventional stone washing with pumice in the treatment of denim fabrics for jeans to remove the indigo dye. WO 94/29510, WO 96/18770, DE 196 12 194 A1 and DE 44 45 088 A1 describe enzymatic delignification processes which use enzymes together with mediators. In general, the disclosed mediators are compounds with the NO—, NOH— or HRNOH structure. These systems, of course, are restricted to use in pulp bleaching. Because the mechanisms underlying lignin-removing pulp bleaching, and this is the process involved here, are entirely different from those underlying the decolorization, removal and/or “destruction” of denim dyes, particularly indigo dyes, in the jeans producing sector, it is entirely surprising that a number of substances of the said NO—, NOH— and HNROH types are also suitable for this application.
WO 97/06244 describes systems for the bleaching of pulp, for dye transfer inhibition and for bleaching stains when used with detergents, which systems employ enzymes (peroxidases, laccases) and enzyme-enhancing (hetero-)aromatic compounds, such as nitroso compounds etc. In this case, as in patents WO 94/12619, WO 94/12620 and WO 94/12621, only the above-described use is intended. The mechanisms of stain decolorization in detergent bleaching or of dye transfer inhibition are entirely different from those underlying the decolorization, removal and/or “destruction” of indigo dyes, as, for example, in denim treatment. Hence, it is quite surprising that a number of substances of the said NO—, NOH— and HNROH-types are also suitable for this application.
Processes are known from said WO 94/12619, WO 94/12620 and WO 94/12621 in which the activity of peroxidase is increased by use of enhancers. Such enhancers are characterized in WO 94/12620 in terms of their half-life. According to WO 94/12621, enhancers are characterized by the formula A=N—N═B where N means nitrogen and A and B are defined cyclic groups. According to WO 94/12621, enhancers are organic chemicals containing at least two aromatic rings of which at least one is substituted with defined groups.
All three applications concern (as already stated) dye transfer inhibition and the use of enhancers together with peroxidases as detergent additives or detergent compositions for washing applications or in cellulose bleaching. The combinations of these enhancers are restricted to use with peroxidases.
Moreover, oxidoreductases, primarily laccases, but also peroxidases, are currently used, mainly for treating denim for jeans.
It is known from patent application WO 96/12846 that laccase and peroxidase+certain enhancers, mainly derivatives of phenothiazine or phenoxazine, are used in two application forms in the treatment of cellulose-containing fabrics, such as cotton, viscose, rayon (artificial silk), ramie, linen, Tencelm, silk or mixtures thereof or mixtures of these fabrics with synthetic fibers, for example a mixture of cotton and spandex (stretch denim), but mainly denim fabrics (mainly for use in jeans).
On the one hand, the system (oxidoreductases+enhancers) is intended as a replacement for the conventional hypochlorite bleaching of denim, usually after a stone washing pretreatment, this enzymatic treatment providing only partial replacement of hypochlorite, because the desired bleaching effect cannot be attained.
On the other hand, the system can be used together with cellulase in stone washing in place of the usual mechanical treatment with pumice, and this represents an improvement over the “treatment with cellulase only”.
The main drawbacks of the system described in WO 96/12846 are the following, among others:
1) To achieve the desired goal, laccase must be used in considerable amounts (about 10 international units [IU]/g of denim);
2) In some cases, optimum treatment requires 2-3 hours;
3) The preferred mediator (here phenothiazine-10-propionic acid) must be used in an amount of about 2 to about 14 mg per gram of denim, which represents a considerable cost;
1) Buffer systems (about 0.1 mol/L) must be used, because otherwise no performance can be achieved, and this also raises the cost of the system. This, for example, is not required for the system of the invention;
2) The color of the enhancer component (long-lived radical) causes “browning” of the fabric.
The general advantage of a laccase and/or oxidoreductase system with enzyme action—enhancing compounds (enhancers, mediators etc.) when used in the above-described treatment of textiles (for example, jeans fabrics) consists, in an improved system over the prior-art systems, in that fashion looks can be achieved, which is not possible with conventional hypochlorite bleaching.
The dyes normally used for jeans denim are vat dyes, such as indigo or indigo derivatives, for example thioindigo, as well as sulfur dyes. By use of such special enzymatic systems, it is possible (as a result of the high specificity of such systems), when a mixed dye system such as an indigo dye and a sulfur dye system is present, to decolorize only the indigo dye, while the sulfur dye is not oxidized. Depending on the enzyme action-enhancing compound used, this can produce almost any desired fabric color (for example, gray shades etc), which is often desirable.
An additional advantage is that the enzymatic treatment is substantially more gentle than bleaching with hypochlorite, and as a result fiber damage is reduced.
In the stone washing process, the ecological effect is of particular importance (in addition to the reduced fiber damage caused by enzymes) considering, for example, that this purely mechanical process produces about 1 kg of stone sludge per kg ofjeans denim.
As can be seen from the prior art, for colored fabrics, in particular, the textile industry has a great need for alternative bleaching processes (alternatives to conventional hypochlorite bleaching) and/or treatment methods as alternatives to stone washing to achieve the bleached look, in the latter case also because of the environmental pollution problems.
The present invention has for an object to minimize or eliminate the drawbacks of the conventional processes: stone washing/bleaching after stone washing or general bleaching of dyed and/or undyed textile fabrics, particularly the pollution problems and fiber damage, as well as the drawbacks of the known oxidoreductase/enhancer systems (for example also NO-radical formation etc) and also the lipase mediated oxidation systems.
Entirely surprisingly, we have now found that the enzymatic ocxidation and/or bleaching systems of the invention exceeds the performance of the aforesaid oxidoreductase-mediator sytems and that it does not exhibit the said drawbacks of the prior art.

DETAILED DESCRIPTION OF THE OXIDATION AND/OR BLEACHING ACCORDING TO THE INVENTION

I) Use in the Beaching of Pulp
The efficacy of the enzymatic oxidation and/or bleaching system of the invention in the modification, degradation or bleaching of lignin, lignin-containing materials or similar substances is often even enhanced when Mg²⁺ ions are present besides the said constituents. The Mg²⁺ ions can be derived, for example, from a salt such as MgSO₄. The Mg²⁺ concentration is in the range from 0.1 to 2 mg/g, and preferably from 0.2 to 0.6 mg/g, of lignin-containing material.
In some cases, the efficacy of the enzyme component system (ECS) of the invention can be increased even further by adding to the system besides Mg²⁺ ions also complexing agents, for example ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), hydroxyethylenediaminetriacetic acid (HEDTA), diethylenetriaminepentamethylenephosphonic acid (DTMPA), nitrilotri-acetic acid (NTA), polyphosphoric acid (PPA) etc. The concentration of said complexing agents is in the range from 0.2 to 5 mg/g, and preferably from 1 to 3 mg/g, of the lignin-containing material. Surprisingly, we have also found that for many wood pulps an acid wash (pH 2 to 6 and preferably 2 to 5) or a Q step (pH 2 to 6 and preferably 2 to 5) preceding the ECS step causes a marked decrease in kappa value compared to processing without this special pretreatment. In the Q step, chelators usually employed for this purpose (for example, EDTA or DTPA) are preferably used at a concentration f rom 0.1 to 1% and particularly from 0.1 to 0.5%.
Reducing agents can be added at the same time, which with the oxidants present give rise to a certain redox potential. Suitable reducing agents are sodium bisulfite, sodium dithionite, ascorbic acid, thio compounds, mercapto compounds or glutathione etc.
It is also possible to add to the system radical generators or radical scavengers (scavenging, for example, OH— or OOH— radicals). These can improve the interaction between the redox and the radical mediators.
Additional metal salt can also be added to the reaction solution. These are important in that they interact with chelators as radical generators or redox centers. In the reaction solution, the salts form cations. Such ions are, among others, Fe²⁺, Fe³⁺, Mn²⁺, Mn³⁺, Mn⁴⁺, Cu¹⁺, Cu²⁺, Ca²⁺, Ti³⁺, Ce⁴⁺ and Al³⁺.
The chelates present in the solution can also serve as mimicking substances for certain oxido-reductases, such as the laccases (copper complexes) or for the lignin or manganese peroxidases (heme complexes). By mimicking substances are meant substances simulating the prosthetic groups of (in the present case) oxidoreductases and, for example, capable of catalyzing oxidation reactions.
Moreover, NaOCl can be added to the reaction mixture. This compound can form singlet oxygen by interacting with hydrogen peroxide.
Finally, it is also possible to use detergents. These include nonionic, anionic, cationic and amphoteric surfactants. Detergents improve the penetration of the enzymes and other components into the fibers.
It may also be necessary to add polysaccharides and/or proteins to the reaction mixture. Suitable polysaccharides are, in particular, glucans, mannans, dextrans, levans, pectins, alginates or vegetable gums, and suitable proteins are gelatins and albumins. These substances serve mainly as protective colloids for the enzymes.
Other proteins that can be added are proteases such as pepsin, bromelain, papain etc. These substances can, among other things, bring about the degradation of extensin (hydroxyproline-rich protein) present in wood, thus improving access to the lignin.
Other suitable protective colloids are amino acids, monosaccharides, oligosaccharides, polyethylene glycol [PEG] types of a wide range of molecular weights, polyethylene oxides, polyethyleneimines and polydimethylsiloxanes.
It is also possible to add to the enzyme component system of the invention substances capable of increasing the hydrophobicity of the reaction mixture, thus bringing about the swelling of the lignin and the fibers and which makes them more susceptible to attack. Such substances are, for example, glycols, such as propylene glycol and ethylene glycol, glycol ethers such as ethylene glycol dimethyl ether etc., and solvents, for example, alcohols such as methanol, ethanol, butanol, amyl alcohol, cyclohexanol, benzyl alcohol and chlorohydrin, phenols such as phenol, methylphenols and methoxyphenols, aldehydes such as formaldehyde and chloral, mercaptans such as butyl mercaptan, benzyl mercaptan and thioglycolic acid, organic acids such as formic, acetic and chloroacetic acid, amines such as ammonia and hydrazine, hydrotropic solvents, for example concentrated solutions of sodium benzoate, other substances such as benzenes, pyridines, dioxane, ethyl acetate, and other basic solvents such as OH⁻/H₂O or OH⁻/alcohol etc.
The process according to the invention can be used not only for the delignification (bleaching) of sulfate, sulfite, organosolv or other wood pulps or lignins, but also for the preparation of wood pulp in general, whether from wood or annual plants, when it is desired to carry out the defibrillation by the usual cooking (digestion) process (possibly combined with mechanical processing or pressure), namely by very gentle digestion, up to kappa numbers in the range from about 50-120 kappa.
In the bleaching as in the preparation of wood pulps, the treatment with the enzymatic system of the invention can be applied once or several times, either before and/or after the washing and extraction of the treated material with NaOH etc., or without these intermediate steps, but also before and/or after pretreatment or post-treatment steps, such as acid washing, Q-steps, alkaline leaching or bleaching steps such as peroxide bleaching, O₂-enhanced peroxide steps, pressurized peroxide steps, O₂-delignification, Cl₂-bleaching, CIO₂-bleaching, Cl₂/CIO₂-bleaching, peracid bleaching, peracid-enhanced O₂/peroxide bleaching, ozone bleaching, dioxirane bleaching, reductive bleaching steps, other treatments such as swelling steps, sulfonations, NO/NO₂treatments, nitrosylsulfuric acid treatment, enzyme treatments, for example treatments with hydrolases, such as cellulases and/or hemicellulases (for example, xylanase, mannase etc) and/or amylases and/or pectinases and/or proteinases and/or lipases and/or amidases and/or oxidoreductases such as, for example, laccases and/or peroxidases etc., or several combined treatments.
This results in substantially further reduced kappa values and substantially increased brightness. Before the enzymatic system treatment, it is also possible to insert an O₂step or, as already mentioned, carry out an acid wash or a Q-step (chelation step).
The invention will be further illustrated by way of the following examples:

EXAMPLE 1

Enzymatic Bleaching with Laccase/Squaric Aid/Acetylaceton
Pulp: Softwood (Sulfate Pulp)
5 g, absolutely dry basis, of wood pulp (O₂-delignified softwood), pulp consistency 30% (about 17 g moist) was added to solutions prepared as follows:
A) To 20 mL of tap water were added 0.1 kg squaric aid and 1 kg acetylaceton per ton pulp with agitation. The pH was adjusted with sulfuric acid and/or sodium hydroxide solution so that, after addition of the wood pulp and the enzyme, the pH was 4.5.
B) To 5 mL of tap water was added this amount of laccase that an activity of 2 IU* per g pulp resulted.
* 1U=conversion of 1 μMol syringaldazine/min/mg solid enzyme
Solutions A and B were combined and diluted to 33 mL. After addition of the wood pulp, the material was mixed in a dough mixer for 2 minutes. The material was then transferred to a reaction vessel preheated to 45° C. and was allowed to incubate 1-4 hours under atmospheric pressure.
The material was washed over a nylon screen (30 μm) and extracted for one hour at 60° C., 2% consistency and using 8% NaOH per gram of wood pulp. The material was again washed after which the kappa number was determined.

EXAMPLE 2

Enzymatic Bleaching With Laccase/4-tert.-butylurazol/acetylaceton
Pulp: Softwood (Sulfate Pulp)
5 g, absolutely dry basis, of wood pulp (O₂-delignified softwood), pulp consistency 30% (about 17 g moist) was added to solutions prepared as follows:
A) To 20 mL of tap water were added 2 kg 4-tert.-butylurazol and 1 kg acetylaceton per ton pulp with agitation. The pH was adjusted with sulfuric acid and/or sodium hydroxide solution so that, after addition of the wood pulp and the enzyme, the pH was 4.5.
B) To 5 mL of tap water was added this amount of laccase that an activity of 2 IU* per g pulp resulted.
* 1U=conversion of 1μMol syringaldazine/min/mg solid enzyme
Solutions A and B were combined and diluted to 33 mL. After addition of the wood pulp, the material was mixed in a dough mixer for 2 minutes. The material was then transferred to a reaction vessel preheated to 45° C. and was allowed to incubate 1-4 hours under atmospheric pressure.
The material was washed over a nylon screen (30 μm) and extracted for one hour at 60° C., 2% consistency and using 8% NaOH per gram of wood pulp. The material was again washed after which the kappa number was determined.

EXAMPLE 3

Enzymatic Bleaching With Laccase/Adipinic Acid Dihydrazide/Acetylaceton
Pulp: Softwood (Sulfate Pulp)
5 g, absolutely dry basis, of wood pulp (O₂-delignified softwood), pulp consistency 30% (about 17 g moist) was added to solutions prepared as follows:
A) To 20 mL of tap water were added 2 kg adipinic acid dihydrazide and 1 kg acetylaceton per ton pulp with agitation. The pH was adjusted with sulfuric acid and/or sodium hydroxide solution so that, after addition of the wood pulp and the enzyme, the pH was 4.5.
B) To 5 mL of tap water was added this amount of laccase that an activity of 2 IU* per g pulp resulted.
* 1U=conversion of 1 μMol syringaldazine/min/mg solid enzyme
Solutions A and B were combined and diluted to 33 mL. After addition of the wood pulp, the material was mixed in a dough mixer for 2 minutes. The material was then transferred to a reaction vessel preheated to 45° C. and was allowed to incubate 1-4 hours under atmospheric pressure.
The material was washed over a nylon screen (30 μm) and extracted for one hour at 60° C., 2% consistency and using 8% NaOH per gram of wood pulp. The material was again washed after which the kappa number was determined.

EXAMPLE 4

Enzymatic Bleaching With Laccase/Hydantoyl-Acetic Acid/Acetylaceton
Pulp: Softwood (Sulfate Pulp)
5 g, absolutely dry basis, of wood pulp (O₂-delignified softwood), pulp consistency 30% (about 17 g moist) was added to solutions prepared as follows:
A) To 20 mL of tap water were added 2 kg hydantoyl-acetic acid and 1 kg acetylaceton per ton pulp with agitation. The pH was adjusted with sulfuric acid and/or sodium hydroxide solution so that, after addition of the wood pulp and the enzyme, the pH was 4.5.
B) To 5 mL of tap water was added this amount of laccase that an activity of 2 IU* per g pulp resulted.
* 1U=conversion of 1 μMol syringaldazine/min/mg solid enzyme
Solutions A and B were combined and diluted to 33 mL. After addition of the wood pulp, the material was mixed in a dough mixer for 2 minutes. The material was then transferred to a reaction vessel preheated to 45° C. and was allowed to incubate 1-4 hours under atmospheric pressure.
The material was washed over a nylon screen (30 μm) and extracted for one hour at 60° C., 2% consistency and using 8% NaOH per gram of wood pulp. The material was again washed after which the kappa number was determined.

EXAMPLE 5

Enzymatic Bleaching With HRP/Dicyandiamide/Acetylaceton
Pulp: Softwood (Sulfate Pulp)
5 g, absolutely dry basis, of wood pulp (O₂-delignified softwood), pulp consistency 30% (about 17 g moist) was added to solutions prepared as follows:
A) To 20 mL of tap water were added 2 kg dicyandiamide and 1 kg acetylaceton per ton pulp with agitation. The pH was adjusted with sulfuric acid and/or sodium hydroxide solution so that, after addition of the wood pulp and the enzyme, the pH was 4.5.
B) To 5 mL of tap water was added 0.1 mg HRP (horseradish peroxidase) per g pulp.
Solutions A and B were combined and diluted to 33 mL. After addition of the wood pulp, the material was mixed in a dough mixer for 2 minutes. The material was then transferred to a reaction vessel preheated to 45° C. and was allowed to incubate 1-4 hours under atmospheric pressure.
The material was washed over a nylon screen (30 μm) and extracted for one hour at 60° C., 2% consistency and using 8% NaOH per gram of wood pulp. The material was again washed after which the kappa number was determined.
EXAMPLE 6
Enzymatic Bleaching With Laccase/Ethylcarbazate/Acetylaceton
Pulp: Softwood (Sulfate Pulp)
5 g, absolutely dry basis, of wood pulp (O₂-delignified softwood), pulp consistency 30% (about 17 g moist) was added to solutions prepared as follows:
A) To 20 mL of tap water were added 3 kg ethylcarbazate and 1 kg acetylaceton per ton pulp with agitation. The pH was adjusted with sulfuric acid and/or sodium hydroxide solution so that, after addition of the wood pulp and the enzyme, the pH was 4.5.
B) To 5 mL of tap water was added this amount of laccase that an activity of 2 IU* per g pulp resulted.
*1U=conversion of 1 μMol syringaldazine/min/mg solid enzyme
Solutions A and B were combined and diluted to 33 mL. After addition of the wood pulp, the material was mixed in a dough mixer for 2 minutes. The material was then transferred to a reaction vessel preheated to 45° C. and was allowed to incubate 1-4 hours under atmospheric pressure.
The material was washed over a nylon screen (30 μm) and extracted for one hour at 60° C., 2% consistency and using 8% NaOH per gram of wood pulp. The material was again washed after which the kappa number was determined.

Claims

1) New enzyme-based process for oxidation and/or bleaching comprising of

oxidoreductases such as laccases and/or peroxidases—seperately or in combination—in the presence of their respective co-substrates like O_2,air, H₂O₂, organic peroxides, peracids etc. and comprising of

enhancer compounds from the class of oxocarbons, from the class of urazoles and hydrazides, from the class of hydantoins and the class of nitril (Cyan)-compounds, and comprising additionally of

carbonyl compounds such as ketones, aldehyds,

whereby the combination of enzyme, co-substrate, enhancer compound and carbonyl compound generate active oxygen species like dioxiranes, dioxetanes, peroxy-compounds etc. or form other reactive compounds or transition states like radicals (kation radicals, anion radicals) or reactive (red/ox-active) neutral compounds as oxidizing and/or bleaching agents.

2) New enzyme-based process for oxidation and/or bleaching according to claim 1 comprising that the enhancer compounds belong to the group of oxocarbons like α-hydroxy-carbonyl compounds, α-dicarbonyl compounds, β-hydroxycarbonyl compounds and β-dicarbonyl compounds, linear compounds with double bonds (enols) and compounds from the group of cyclic oxocarbons like deltic acid, squaric acid, crocoic acid, rhodizonic acid, tetrahydroxy-p-hydroquinone and their salts and derivatives.

3) New enzyme-based process for oxidation and/or bleaching according to claim 1 comprising that that the enhancer compounds belong to the group of amides such as hydrazides, cyclic hydrazides, urazoles and phthalhydrazides.

4) New enzyme-based process for oxidation and/or bleaching according to claim 1 comprising that that the enhancer compounds belong to the group of imides like hydantoins, cyclic imides and hydantoin derivatives.

5) New enzyme-based process for oxidation and/or bleaching according to claim 1

5) New enzyme-based process for oxidation and/or bleaching according to claim 1 comprising that that the enhancer compounds belong to the group of nitril-(Cyan) compounds such as cyanamide or dicyandiamide.

6) New enzyme-based process for oxidation and/or bleaching according to claim 1 comprising that additionally carbonyl compounds—mainly non-cyclic substances—are added.

7) New enzyme-based process for oxidation and/or bleaching according to claim 1 comprising that as enzymes oxidoreductases of the classes 1.1.1. bis 1.97 are used.

8) New enzyme-based process for oxidation and/or bleaching according to claim 1 comprising that as oxidoreductases laccases and peroxidases are used.

9) New enzyme-based process for oxidation and/or bleaching according to claim 1 comprising that as additional enzyme-based systems HOS (hydrolase mediated oxidation system), peroxynitreous acid generating system, ferrocene/peroxide system, organosulfonic acid/peroxide/ketone system, activated sulfite/superoxide/ketone system and other state-of-the-art oxidoreductase mediator systems are added either simultaneously or successively in any order are added.

10) New enzyme-based process for oxidation and/or bleaching according to claim 1 comprising that as co-substrates preferrably air, oxygen, ozone, peroxides, such as H₂O₂, organic peroxides, peracids such as peracetic, performic, persulfuric, pernitric, metachloroperoxybenzoic and perchloric acid, per compounds such as perborates, percarbonates or persulfates, or oxygen species and the radicals thereof such as the OH, OOH⁻ and OH⁺ radical, superoxide (O₂—), dioxygenyl cation (O₂ ⁺), singlet oxygen are used.

11) Use of the enzymatic oxidation and/or bleaching system according to claim 1 to 10 in a process for the delignification and/or modificaton and/or bleaching of pulps from wood and annual plants and high yield pulps and deinked pulps, whereby the reactions of the oxidation and/or bleaching systems are performed in a pH-range of pH 3 to 11, preferably pH 3 to 9, at a temperature of 20 to 95° C., preferably of 40 to 95° C., at a consistency of 0.5 to 40%, preferably of 4 to 15%, in the presence of oxygen or air at atmospheric pressure to a slight overpressure (up to 2 bar). The laccase concentrations or peroxidase concentrations lay in the range of 2-500 g pure enzyme per ton pulp, the concentrations of the enhancer compounds lay in the range of 1-15 kg per ton pulp, the H₂O₂concentrations in the range of 0.2 to 15 kg per ton pulp and the concentrations of carbonyl compounds in the range of 0.2 to 5 kg per ton pulp.

12) Use of the enzymatic oxidation and/or bleaching system according to claim 1 to 11 in a process for the delignification and /or modificaton and/or bleaching of pulps from wood and annual plants and high yield pulps and deinked pulps whereby an acid wash or a Q-step is used before and/or after the reaction of the enzyme component system and the acid wash is carried out at 60-120° C., at pH 2 to 5.5, for 30-90 min and at 4-20% pulp consistency, and the Q-step is carried out with 0.05-1%, preferably with 0.2 to 0.5% of chelator at 60-100° C., at pH 2 to 5.5 for 30-90 min and a consistency of 4-20%.

13) Use of the enzymatic oxidation and/or bleaching system according to claim 1 to 12 in a process for the delignification and /or modificaton and/or bleaching of pulps from wood and annual plants and high yield pulps and deinked pulps, whereby the acid wash and the Q-step are carried out for 1 hour at 60-90° C., at pH 2 to 5 and at 10% pulp consistency.

14 Use of the enzymatic oxidation and/or bleaching system according to claim 1 to 13 in a process for the delignification and/or modificaton and/or bleaching of pulps from wood and annual plants and high yield pulps and recycling pulps whereby it can be applied once or several times, either before and/or after the washing and extraction of the treated material with NaOH etc., or without these intermediate steps, but also before and/or after pretreatment or post-treatment steps, such as acid washing, Q-steps, alkaline leaching or bleaching steps such as peroxide bleaching, O₂-enhanced peroxide steps, pressurized peroxide steps, O₂-delignification, Cl₂-bleaching, CIO₂-bleaching, Cl₂/CIO₂-bleaching, peracid bleaching, peracid-enhanced O₂/peroxide bleaching, ozone bleaching, dioxirane bleaching, reductive bleaching steps, other treatments such as swelling steps, sulfonations, NO/NO₂treatments, nitrosylsulfuric acid treatment, enzyme treatments, for example treatments with hydrolases, such as cellulases and/or hemicellulases (for example, xylanase, mannase etc) and/or amylases and/or pectinases and/or proteinases and/or lipases and/or amidases and/or oxidoreductases such as, for example, laccases and/or peroxidases etc., or several combined treatments.

15) Use of the enzymatic oxidation and/or bleaching system according to claim 1 to 14 in a process for the delignification and /or modificaton and/or bleaching of pulps from wood and annual plants and high yield pulps and deinked pulps, said process being carried out in several steps and whereby between each step is applied a washing or washing and extraction step with alkaline hydroxide solution, or neither washing nor extraction takes place.

16) Use of the enzymatic oxidation and/or bleaching system according to claim 1 bis 15 in the bleaching of cellulose/wood pulp, in the treatment of different wastewaters, in the preparation of lignin solutions or gels of the corresponding binders/adhesives and of wood-based composites, as enzymatic deinking system, as oxidation systems in organic synthesis, as bleaching agent in detergents, as bleaching and/or oxidation systems in textile industry, in coal liquefaction.