DE10132529A1 - Culture medium for the cultivation of filamentous fungi - Google Patents

Abstract

The invention relates to a culture medium for cultivating filamentous fungi and to a method for obtaining enzymes from filamentous fungi.

Description

The present invention relates to a culture medium for cultivation filamentous mushrooms and a method for obtaining enzymes from filamentous ones Mushrooms.

Filamentous fungi are biotechnologically important organisms, one Synthesize a variety of different enzymes. However, is problematic often the cultivation of the mushrooms.

From international laid-open documents WO-A2-99 / 49020 and WO-A2- 99/49010 is the extraction and purification of phenol oxidizing enzymes Stachybotrys species known. About the culture media used however, no further details were given.

KOROLJOVA-SKOROBOGAT'KO et al. describe in biotechnol. Appl. Biochem (1998), 28, pp. 47-54, the purification and characterization of a laccase the filamentous mushroom Coriolus hirsutus. The culture medium is on p. 48, left Column, described in detail below. Find it on page 50, right column, above examples of different inducers of the biosynthesis of laccase.

However, attempts by the applicant have shown that they are suitable for cultivation filamentous fungi previously available culture media both regarding the growth of the mushrooms, as well as regarding the enzyme yield are satisfactory.

Surprisingly, it was found that a significant reduction in the concentration of CuSO ₄ .5H ₂ O compared to that of KOROLJOVASKOROBOGAT'KO et al. describe concentration of 0.25 g / L leads to an acceleration of the growth of filamentous fungi.

• a) etwa 5 bis etwa 20 [g/L] Glucose,
• b) etwa 1 bis etwa 6 [g/L] Peptone,
• c) etwa 0,006 bis etwa 0,03 [g/L] CuSO₄ enthält.

The present invention therefore relates to a culture medium for the cultivation of filamentous fungi which

• a) about 5 to about 20 [g / L] glucose,
• b) about 1 to about 6 [g / L] peptones,
• c) contains about 0.006 to about 0.03 [g / L] CuSO ₄ .

• a) etwa 5 bis etwa 20 [g/L] Glucose,
• b) etwa 1 bis etwa 6 [g/L] Peptone,
• c) etwa 0,006 bis etwa 0,03 [g/L] CuSO₄,
• d) etwa 0,01 bis etwa 1,0 [g/L] KH₂PO₄,
• e) etwa 0,0003 bis etwa 0,003 [g/L] ZnSO₄,
• f) etwa 0,01 bis etwa 0,8 [g/L] K₂HPO₄,
• g) etwa 0,00005 bis etwa 0,005 [g/L] FeSO₄,
• h) etwa 0,009 bis etwa 0,09 [g/L] MnSO₄ sowie
• i) etwa 0,005 bis etwa 0,5 [g/L] MgSO₄.

The culture medium preferably contains

• a) about 5 to about 20 [g / L] glucose,
• b) about 1 to about 6 [g / L] peptones,
• c) about 0.006 to about 0.03 [g / L] CuSO ₄ ,
• d) about 0.01 to about 1.0 [g / L] KH ₂ PO ₄ ,
• e) about 0.0003 to about 0.003 [g / L] ZnSO ₄ ,
• f) about 0.01 to about 0.8 [g / L] K ₂ HPO ₄ ,
• g) about 0.00005 to about 0.005 [g / L] FeSO ₄ ,
• h) about 0.009 to about 0.09 [g / L] MnSO ₄ and
• i) about 0.005 to about 0.5 [g / L] MgSO ₄ .

• a) 7 bis 18 [g/L] Glucose,
• b) 2 bis 5 [g/L] Peptone,
• c) 0,009 bis 0,022 [g/L] CuSO₄,
• d) 0,1 bis 1,0 [g/L] KH₂PO₄,
• e) 0,0004 bis 0,002 [g/L] ZnSO₄,
• f) 0,02 bis 0,6 [g/L] K₂HPO₄,
• g) 0,0001 bis 0,005 [g/L] FeSO₄,
• h) 0,02 bis 0,07 [g/L] MnSO₄ sowie
• i) 0,01 bis 0,4 [g/L] MgSO₄.

The culture medium contains particularly preferably

• a) 7 to 18 [g / L] glucose,
• b) 2 to 5 [g / L] peptones,
• c) 0.009 to 0.022 [g / L] CuSO ₄ ,
• d) 0.1 to 1.0 [g / L] KH ₂ PO ₄ ,
• e) 0.0004 to 0.002 [g / L] ZnSO ₄ ,
• f) 0.02 to 0.6 [g / L] K ₂ HPO ₄ ,
• g) 0.0001 to 0.005 [g / L] FeSO ₄ ,
• h) 0.02 to 0.07 [g / L] MnSO ₄ and
• i) 0.01 to 0.4 [g / L] MgSO ₄ .

• a) 10 bis 15 [g/L] Glucose,
• b) 3 [g/L] Peptone,
• c) 0,016 [g/L] CuSO₄,
• d) 0,6 [g/L] KH₂PO₄,
• e) 0,0006 [g/L] ZnSO₄,
• f) 0,04 bis 0,4 [g/L] K₂HPO₄,
• g) 0,0003 bis 0,003 [g/L] FeSO₄,
• h) 0,04 [g/L] MnSO₄ sowie
• i) 0,02 bis 0,2 [g/L] MgSO₄.

The culture medium contains very particularly preferably

• a) 10 to 15 [g / L] glucose,
• b) 3 [g / L] peptones,
• c) 0.016 [g / L] CuSO ₄ ,
• d) 0.6 [g / L] KH ₂ PO ₄ ,
• e) 0.0006 [g / L] ZnSO ₄ ,
• f) 0.04 to 0.4 [g / L] K ₂ HPO ₄ ,
• g) 0.0003 to 0.003 [g / L] FeSO ₄ ,
• h) 0.04 [g / L] MnSO ₄ and
• i) 0.02 to 0.2 [g / L] MgSO ₄ .

• - das Kupfer(II)sulfat als CuSO₄.5H₂O,
• - das Zinksulfat als ZnSO₄.7H₂O,
• - das Eisen(II)sulfat als FeSO₄.7H₂O,
• - das Mangansulfat als MnSO₄.H₂O und
• - das Magnesiumsulfat als MgSO₄.7H₂O

eingesetzt wird. It is preferred according to the invention if

• the copper (II) sulfate as CuSO ₄ .5H ₂ O,
• the zinc sulfate as ZnSO ₄ .7H ₂ O,
• - the iron (II) sulfate as FeSO ₄ .7H ₂ O,
• - The manganese sulfate as MnSO ₄ .H ₂ O and
• - the magnesium sulfate as MgSO ₄ .7H ₂ O

is used.

• a) ABTS®
• b) 3,4-Xylidin,
• c) Huminsäuren,
• d) Kaffeesäure,
• e) Guajakol,
• f) Sinapinsäure,
• g) o-Toluidin,
• h) chelatisierenden Ligninverbindungen,
• i) Syringaldazin und vorzugsweise
• j) Benzylalkohol.

In a preferred embodiment of the invention, the culture medium additionally contains a compound which induces the biosynthesis of enzymes, in particular one of the compounds which are mentioned in WO 99/49020, p. 17, lines 4 to 11. Reference is hereby expressly made to this document. Further preferred compounds as inductors are selected from:

• a) ABTS®
• b) 3,4-xylidine,
• c) humic acids,
• d) caffeic acid,
• e) guaia cole,
• f) sinapic acid,
• g) o-toluidine,
• h) chelating lignin compounds,
• i) syringaldazine and preferably
• j) benzyl alcohol.

According to the invention, the inductors are used in an amount of 1 nmol / l to 100 mmol / l, in particular from 100 nmol / l to 20 mmol / l.

ABTS® and benzyl alcohol are particularly preferred according to the invention. In a preferred embodiment is ABTS® as an inductor in an amount of 0.5 up to 1.5 mmol / l used. In a likewise preferred embodiment Benzyl alcohol is used in an amount of 5 to 10 mmol / l.

These compounds are particularly suitable for the synthesis of Oxidoreductases, especially of phenol oxidizing enzymes (phenol oxidases) induce. If other enzymes are to be obtained more intensely, can corresponding other inductors known to the person skilled in the art Culture medium can be added.

• a) filamentöse Pilze in einem erfindungsgemäßen Kulturmedium kultiviert und
• b) die Enzyme aus den kultivierten Pilzen bzw. aus dem Kulturmedium isoliert.

Another object of the present invention is a process for the production of enzymes from filamentous fungi, which is characterized in that

• a) cultivated filamentous fungi in a culture medium according to the invention and
• b) the enzymes are isolated from the cultivated fungi or from the culture medium.

The method according to the invention is basically with all filamentous Fungi feasible.

For example, those in the master list of the DSMZ (German Strain collection of microorganisms and cell cultures GmbH, Braunschweig) Organisms listed on the Internet at the address (http: / / www.dsmz.de/species/fungi.htm) is visible.

The fungi of the following genera are suitable according to the invention:
Absidia, Acremoniella, Acremonium, Actinomucor, Agaricus, Agrocybe, Aleurodiscus, Allomyces, Alternaria, Amanita, Amauroascus, Ambrosiella, Ampelomyces, Amphiporthe, Amylostereum, Anellaria, Anixiella, Anixiopsis, Anthracobia, Apropia, Aphrodisia Arachniotus, Armillaria, Arthrinium, Arthrobotrys, Arthroderma, Ascobolus, Ascochyta, Ascocoryne, Ascodesmis, Ascodichaena, Ascoidea, Ascosphaera, Ashbya, Aspergillus, Asterophora, Athelia, Aulographina, Aurantasasiduxidium, Aurobasipidium, Aurobia Aid, Aureobasidium Aurobasidium, Aurobiaus Aurobasidium, Aurobia Blakeslea, Blastobotrys, Boletus, Bombardia, Botryobasidium, Botryodiplodia, Botryosphaeria, Botryotinia, Botryotrichum, Botrytis, Brunchorstia, Byssochlamys, Calcarisporium, Caldariomyces, Calocera, Calonectria, Cantharellocisis, Cocellociumsporium, Cenocciumsporium, Cenangium Porc, Cenangium Porcupine Ceuthospora, Chaetocladium, Chaetomidium, Chaetomium, Chalara, Chalciporus, Choanephora, Chondrostereum, Chrysosporium, Circinella, Cladobotryum, Cladorrhinum, Cladosporium, Claviceps, Climacocystis, Climacodon, Clitocybe, Clitocybula, Clitopilus, Cochliobolus, Colemetiaophobia, Colomiaiaiophobia, Colophobia Coniothyrium, Copelandia, Coprinus, Cordana, Cordyceps, Coriolopsis, Coriolus, Corticium, Coryne, Corynespora, Crinipellis, Crucibulum, Cryphonectria, Cryptodiaporthe, Cryptosporiopsis, Cunninghamella, Curvularia, Cyathus, Cylindrocemaphumphum, Cylindrocemaidium, Cylindrocemaidum Dacrymyces, Dactylaria, Dactylium, Daedalea, Daedaleopsis, Datronia, Delacroixia, Dendryphion, Dermea, Diaporthe, Dichomitus, Dictyopanus, Dictyosporium, Dictyostelium, Didymella, Diheterospora, Diplodia, Dipisascopia, Dipodascodia, Dipodascodia, Dipodascodia, Dipodascodia, Dipodascki, Dipodascodia, Dipodascodia, Dipodascodia, Dipodascodia, Dipodascodia, Dipodascki Dothiorella, Drechslera, Drepanopeziza, Durella, Eladia, E leutherascus, Embellisia, Emericella, Emericellopsis, Endoconidiophora, Endothia, Entomophthora, Epicoccum, Epidermophyton, Eremascus, Eremothecium, Eupenicillium, Eurasina, Eurotium, Exidia, Exobasidium, Exophiala, Fomyellumisulumina, Fserellumumisulumina, Fserellumumisulumina, Fserellum, Fomyellum Fusarium, Fusicoccum, Fusidium, Gaeumannomyces, Ganoderma, Gelasinospora, Geomyces, Geotrichum, Gerronema, Gibberella, Gilmaniella, Glenospora, Gliocladium, Gloeocystidiellum, Gloeophyllum, Gloeosporium, Glomerella, Gnomonia, Gongronella, Graphium, Gremmeniella, Grifola, Guignardia, Gymnascella, Gymnoascus, Gymnopilus, Gyromitra, Hamigera, Harzia, Hebeloma, Helicostylum, Helminthosporium, Helotium, Hendersonia, Hercospora, Hericium, Heterobasidion, Hirneola, Hohenbuehelia, Hormoconis, Hormodendrum, Humicola, Hyalodendron, Hydnopolusus, Hyoprophosus, Hydrophysus Hypochnus, Hypocrea, Hypomyces, Hypoxylon, Hypsizygus, Inonotus, Irpex , Isaria, Kabatiella, Kabatina, Kuehneromyces, Laccaria, Lachnellula, Laetiporus, Laricifomes, Laxitextum, Lecythophora, Lentinula, Lentinus, Lentodium, Lenzites, Lepiota, Lepista, Leptographium, Leptoporus, Leptosphaerocarominus, Leucophylocarpus, Leucophobia, Leucophobia , Lycoperdon, Lyophyllum, Macrohyporia, Macrolepiota, Macrophomina, Malbranchea, Marasmius, Margaritispora, Marsonnina, Martininia, Megacollybia, Melanomma, Melanoporia, Memnoniella, Meripilus, Merismodes, Merulius, Metarhician, Microascus, Microbodochroumium, Microbodochroumum, Microbodochroium, Microbotochroum, Microbotochium, Microbotochium, Microbotochium, Microbotochium, Microbotochium, Microbotochium, Microbotochium, Microbotochium, Microbotochium, Microbotochium, Microbotochium, Microbotochium, Microbotochium, Microbotochium, Microbotochium , Monilia, Moniliella, Monilinia, Monodictys, Monostichella, Morchella, Mortierella, Mucor, Mutinus, Myceliophthora, Mycena, Mycocentrospora, Mycogone, Mycosphaerella, Mycotypha, Myrothecium, Naemacyclus, Naemospora, Niaocosoloma, Nauocosomoma, Nauocosoloma, Nauocosolomium , Neurospora, Nigrospora, Nothopanus, Oedocephalum, Oligoporus, Omphalotus , Onnia, Oospora, Ophiostoma, Osmoporus, Oudemanstella, Paecilomyces, Panaeolina, Panaeolus, Panellus, Panus, Papulaspora, Parasitella, Paxillus, Pellicularia, Penicilliopsis, Penicillium, Peniophora, Periconia, Pezellaotiopsiotomizellais, Roma , Phacidium, Phaeolus, Phallus, Phanerochaete, Phellinus, Phialophora, Phlebia, Phlebiopsis, Pholioata, Pholiota, Pholiotina, Phoma, Phomopsis, Phycomyces, Phytophthora, Pilaira, Pilobolus, Piloderma , Pleurotellus, Pleurotus, Plicatura, Podospora, Pollacia, Polyporus, Polysphondylium, Polystictus, Poria, Poronia, Potebniamyces, Preussia, Prototheca, Pseudoarachniotus, Pseudoclitocybe, Pseudohansfordia, Pseudonymia, Pyythocnomia, Pseudonectria, Pseudonectria, Pseudonectria, Pseudonectria, Pseudonectria, Pseudonectria, Pseudonectria, Pseudonectria, Pseudonectria, Pseudonectria, Pseudonectria, Pseudonectria, Pseudonectria, Pseudonectria, Pseudonectria, Pseudonectria, Pseudonectria, Pseudonectria, Pseudonectria, Pythonyma , Pythium, Radiomyces, Raffaelea, Ramaria, Ramichloridium, Resinicium, Rhinocladiella, Rhizoctoni a, Rhizomucor, Rhizopus, Rhizosphaera, Rutstroemia, Ryparobius, Saprolegnia, Sarcodontia, Schizonella, Schizophyllum, Schizopora, Sclerophoma, Sclerotinia, Sclerotium, Scopulariopsis, Scytalidium, Sepedonium, Sporridium, Sibomyaproma, Sibomyaproma, Sibomyaproma Spicaria, Spiniger, Spondylocladium, Spongipellis, Spongiporus, Sporormiella, Sporothrix, Sporotrichum, Stachybotrys, Staurophoma, Stemphylium, Stereum, Sterigmatocystis, Stictis, Strasseria, Stromatinia, Stropharia, Stysanophoria, Symaproma, Horophysalphomostomy, Synodroma, Sinusphroma, Synillusaroma, Synodroma, Sinusphromus Tapinella, Tephrocybe, Termitomyces, Thamnidium, Thamnostylum, Thanatephorus, Thelebolus, Thermoascus, Thermomyces, Thielavia, Thielaviopsis, Thysanophora, Tiarosporella, Tieghemella, Tieghemiomyces, Tilachlidium, Trillladium, Trillladium, Trillladium, Tilletopsis, Tilletopsis, Tilletopsis, Tilletopsis, Tilletopsis, Trilletium, Trilletium, Trilletium, Tilletopsis, Tilletopsis, Tilletopsis, Trilli Tricholoma, Trichophyton, Trichosporon, Trichothecium, Trichu rus, Tritirachium, Truncatella, Tyromyces, Ulocladium, Urocystis, Ustilago, Valsa, Venturia, Verpa, Verticillium, Volutella, Volvariella, Vuilleminia, Wallemia, Westerdykella, Wojnowicia, Wolfiporia, Xenomeris, Xerocomus, Xgorulaynchylus, Xerulaogcharia

Particularly suitable for carrying out the method according to the invention Mushrooms are selected from Genera Acremonium, Aspergillus, Coriolus, Coprinus, Pleurotus, Stachybotrys and Fusarium, especially under Mushrooms of the genus Stachybotrys, preferably among mushrooms of the species Stachybotrys atra, Stachybotrys aurantia, Stachybotrys bisbyi, Stachybotrys chartarum, Stachybotrys parvispora, Stachybotrys kampalensis, Stachybotrys theobromae, Stachybotrys cylindrospora, Stachybotrys dichroa, Stachybotrys oenanthes and Stachybotrys nilagerica, particularly preferred among Stachybotrys bisbyi and Stachybotrys chartarum.

• a) Proteasen,
• b) Oxidoreductasen, wie Oxidasen, insbesondere Phenoloxidasen, Peroxidasen, Laccasen, Tyrosinasen;
• c) Lipasen und
• d) Glycosidasen, wie Amylasen, Cellulasen und Glucanasen.

The method according to the invention is particularly suitable for the production of enzymes which are selected from

• a) proteases,
• b) oxidoreductases, such as oxidases, in particular phenol oxidases, peroxidases, laccases, tyrosinases;
• c) lipases and
• d) glycosidases such as amylases, cellulases and glucanases.

The enzymes are isolated in a manner known to those skilled in the art, as described, for example, in the following references, to which reference is hereby expressly made:
Gulati R, Saxena RK, Gupta R; Fermentation and downstream processing of lipase from Aspergillus terreus; PROCESS BIOCHEM 36 (1-2): 149-155 SEP 2000.

Maheshwari R, Bharadwaj G, Bhat MK; Thermophilic fungi: Their physiology and enzymes; MICROBIOL MOL BIOL R 64 (3): 461- + SEP 2000.

Dunaevsky YE, Gruban TN, Beliakova GA, et al .; Enzymes secreted by filamentous fungi: Regulation of secretion and purification of an extracellular protease of Trichoderma harzianum; BIOCHEMISTRY-MOSCOW + 65 (6): 723-727 JUN 2000.

Conesa A, from the Hondel CAMJJ, Punt PJ; Studies on the production of fungal peroxidases in Aspergillus niger; APPL ENVIRON MICROB 66 (7): 3016-3023 JUL 2000.

Dalboge H, Lange L; Using molecular techniques to identify new microbial Biocatalysts; TRENDS BIOTECHNOL 16 (6): 265-272 JUN 1998.

Maia MMD, Heasley A, de Morais MMC, et al .; Effect of culture conditions on lipase production by Fusarium solani in batch fermentation; Bioresource TECHNOL 76 (1): 23-27 JAN 2001.

Kawasaki L, Aguirre J; Multiple catalase genes are differentially regulated in Aspergillus nidulans; J BACTERIOL 183 (4): 1434-1440 FEB 2001

Another object of the present invention is the use of a Culture medium according to the invention for the cultivation of filamentous fungi.

Another object of the present invention is the use of immediate process products from one of the invention Processes in detergents, cleaning agents, hand washing agents, Hand dishwashing detergents, machine dishwashing detergents, cosmetic or pharmaceutical Preparations, hair tinting and coloring agents and coloring agents or bleaching textiles, pulp, paper, furs, skins or leather.

Other independent objects of the present invention are detergents, cleaning agents, hand washing agents, hand dishwashing detergents, Dishwasher detergents, cosmetic or pharmaceutical Preparations, hair tinting and hair dyeing and dyeing or bleaching preparations of textiles, pulp, paper, furs, skins or leather containing immediate Process products from one of the processes according to the invention.

In the preferred case according to the invention of a hair tint or Hair dye is the immediate process product preferably a phenol oxidase.

The enzyme can be incorporated into the colorant itself; is preferred but it is made up separately from the dye precursors and only added to the colorant immediately before use.

The enzyme is preferably used in an amount of 0.001-1% by weight, based on the amount of protein of the enzyme and the entire colorant used.

• - Oxidationsfarbstoffvorprodukte vom Entwickler- und/oder Kuppler-Typ, und
• - Vorstufen naturanaloger Farbstoffe, wie Indol- und Indolin-Derivate, sowie Mischungen von Vertretern dieser Gruppen enthalten.

The present invention is not subject to any restrictions with regard to the dye precursors used in the colorants according to the invention. The colorants of the invention can be used as dye precursors

• - Oxidation dye precursors of the developer and / or coupler type, and
• - Contain precursors of nature-analogous dyes, such as indole and indoline derivatives, and mixtures of representatives of these groups.

Primary aromatic amines are usually used as developer components with another, free or in para or ortho position substituted hydroxy or amino group, diaminopyridine derivatives, heterocyclic Hydrazones, 4-aminopyrazole derivatives and 2,4,5,6-tetraaminopyrimidine and its derivatives used.

• - G¹ steht für ein Wasserstoffatom, ein C₁- bis C₄-Alkylradikal, ein C₁- bis C₄-Monohydroxyalkylradikal, ein C₂- bis C₄-Polyhydroxyalkylradikal, ein (C₁- bis C₄)-Alkoxy-(C₁- bis C₄)-alkylradikal, ein 4'-Aminophenylradikal oder ein C₁- bis C₄-Alkylradikal, das mit einer stickstoffhaltigen Gruppe, einem Phenyl- oder einem 4'-Aminophenylrest substituiert ist;
• - G² steht für ein Wasserstoffatom, ein C₁- bis C₄-Alkylradikal, ein C₁- bis C₄-Monohydroxyalkylradikal, ein C₂- bis C₄-Polyhydroxyalkylradikal, ein (C₁- bis C₄)-Alkoxy-(C₁- bis C₄)-alkylradikal oder ein C₁- bis C₄- Alkylradikal, das mit einer stickstoffhaltigen Gruppe substituiert ist;
• - G³ steht für ein Wasserstoffatom, ein Halogenatom, wie ein Chlor-, Brom, Jod- oder Fluoratom, ein C₁- bis C₄-Alkylradikal, ein C₁- bis C₄-Monohydroxyalkylradikal, ein C₁- bis C₄-Hydroxyalkoxyradikal, ein C₁- bis C₄-Acetylaminoalkoxyradikal, ein C₁- bis C₄- Mesylaminoalkoxyradikal oder ein C₁- bis C₄-Carbamoylaminoalkoxyradikal;
• - G⁴ steht für ein Wasserstoffatom, ein Halogenatom oder ein C₁- bis C₄- Alkylradikal oder
• - wenn G³ und G⁴ in ortho-Stellung zueinander stehen, können sie gemeinsam eine verbrückende α,ω-Alkylendioxogruppe, wie beispielsweise einen Ethylendioxygruppe bilden.

It may be preferred according to the invention to use a p-phenylenediamine derivative or one of its physiologically tolerable salts as the developer component. P-Phenylenediamine derivatives of the formula (I) are particularly preferred


in which

• - G ¹ represents a hydrogen atom, a C ₁ to C ₄ alkyl radical, a C ₁ to C ₄ monohydroxyalkyl radical, a C ₂ to C ₄ polyhydroxyalkyl radical, a (C ₁ to C ₄ ) alkoxy (C ₁ to C ₄ ) alkyl radical, a 4'-aminophenyl radical or a C ₁ to C ₄ alkyl radical which is substituted by a nitrogen-containing group, a phenyl or a 4'-aminophenyl radical;
• G ² represents a hydrogen atom, a C ₁ to C ₄ alkyl radical, a C ₁ to C ₄ monohydroxyalkyl radical, a C ₂ to C ₄ polyhydroxyalkyl radical, a (C ₁ to C ₄ ) alkoxy (C ₁ to C ₄ ) alkyl radical or a C ₁ to C ₄ alkyl radical substituted with a nitrogen-containing group;
• - G ³ represents a hydrogen atom, a halogen atom, such as a chlorine, bromine, iodine or fluorine atom, a C ₁ to C ₄ alkyl radical, a C ₁ to C ₄ monohydroxyalkyl radical, a C ₁ to C ₄ -Hydroxyalkoxyradikal, a C ₁ - to C ₄ -Acetylaminoalkoxyradikal, a C ₁ - to C ₄ - Mesylaminoalkoxyradikal or a C ₁ - to C ₄ -Carbamoylaminoalkoxyradikal;
• G ⁴ represents a hydrogen atom, a halogen atom or a C ₁ to C ₄ alkyl radical or
• - If G ³ and G ⁴ are ortho to each other, they can together form a bridging α, ω-alkylenedioxo group, such as an ethylenedioxy group.

Examples of the C ₁ - to C ₄ -alkyl radicals mentioned as substituents in the compounds according to the invention are the groups methyl, ethyl, propyl, isopropyl and butyl. Ethyl and methyl are preferred alkyl radicals. C ₁ to C ₄ alkoxy radicals preferred according to the invention are, for example, a methoxy or an ethoxy group. Further preferred examples of a C ₁ - to C ₄ -hydroxyalkyl group are a hydroxymethyl, a 2-hydroxyethyl, a 3-hydroxypropyl or a 4-hydroxybutyl group. A 2-hydroxyalkyl group is particularly preferred. Examples of halogen atoms according to the invention are F, Cl or Br atoms, Cl atoms are very particularly preferred. According to the invention, the other terms used are derived from the definitions given here. Examples of nitrogen-containing groups of the formula (II) are in particular the amino groups, C ₁ to C ₄ monoalkylamino groups, C ₁ to C ₄ dialkylamino groups, C ₁ to C ₄ trialkylammonium groups, C ₁ to C ₄ monohydroxyalkylamino groups, Imidazolinium and ammonium.

Particularly preferred p-phenylenediamines of the formula (I) are selected from p-phenylenediamine, p-toluenediamine, 2-chloro-p-phenylenediamine, 2,3-dimethyl p-phenylenediamine, 2,6-dimethyl-p-phenylenediamine, 2,6-diethyl-p- phenylenediamine, 2,5-dimethyl-p-phenylenediamine, N, N-dimethyl-p- phenylenediamine, N, N-diethyl-p-phenylenediamine, N, N-dipropyl-p- phenylenediamine, 4-amino-3-methyl- (N, N-diethyl) -aniline, N, N-bis- (β- Hydroxyethyl) -p-phenylenediamine, 4-N, N-bis- (β-hydroxyethyl) amino-2- methylaniline, 4-N, N-bis- (β-hydroxyethyl) amino-2-chloroaniline, 2- (β-hydroxyethyl) - p-phenylenediamine, 2-fluoro-p-phenylenediamine, 2-isopropyl-p-phenylenediamine, N- (β-hydroxypropyl) -p-phenylenediamine, 2-hydroxymethyl-p-phenylenediamine, N, N- Dimethyl-3-methyl-p-phenylenediamine, N, N- (ethyl, -β-hydroxyethyl) -p- phenylenediamine, N- (β, γ-dihydroxypropyl) -p-phenylenediamine, N- (4'- Aminophenyl) -p-phenylenediamine, N-phenyl-p-phenylenediamine, 2- (β- Hydroxyethyloxy) -p-phenylenediamine, 2- (β-acetylaminoethyloxy) -p- phenylenediamine, N- (β-methoxyethyl) -p-phenylenediamine and 5,8-diaminobenzo 1,4-dioxane and its physiologically tolerable salts.

According to the invention, p-phenylenediamine derivatives which are particularly preferred Formula (I) are p-phenylenediamine, p-toluenediamine, 2- (β-hydroxyethyl) -p- phenylenediamine and N, N-bis (β-hydroxyethyl) -p-phenylenediamine.

According to the invention, it can also be preferred as the developer component To use compounds which contain at least two aromatic nuclei, which are substituted with amino and / or hydroxyl groups.

• - Z¹ und Z² stehen unabhängig voneinander für ein Hydroxyl- oder NH₂- Radikal, das
• - gegebenenfalls durch ein C₁- bis C₄-Alkylradikal, durch ein C₁- bis C₄- Hydroxyalkylradikal und/oder durch eine Verbrückung Y substituiert ist,
• - die Verbrückung Y steht für eine Alkylengruppe mit 1 bis 14 Kohlenstoffatomen,
• - wie beispielsweise eine lineare oder verzweigte Alkylenkette oder einen Alkylenring, die von einer oder mehreren stickstoffhaltigen Gruppen und/oder einem oder mehreren Heteroatomen wie Sauerstoff-, Schwefel- oder Stickstoffatomen unterbrochen oder beendet sein kann und eventuell durch ein oder mehrere Hydroxyl- oder C₁- bis C₈-Alkoxyradikale substituiert sein kann,
• - G⁵ und G⁶ stehen unabhängig voneinander für ein Wasserstoff- oder Halogenatom,
• - ein C₁- bis C₄-Alkylradikal, ein C₁- bis C₄-Monohydroxyalkylradikal, ein C₂- bis C₄- Polyhydroxyalkylradikal, ein C₁- bis C₄-Aminoalkylradikal oder eine direkte Verbindung zur Verbrückung Y,
• - G⁷, G⁸, G⁹, G¹⁰, G¹¹ und G¹² stehen unabhängig voneinander für ein Wasserstoffatom, eine direkte Bindung zur Verbrückung Y oder ein C₁- bis C₄- Alkylradikal,
  mit der Maßgabe, dass die Verbindungen der Formel (II) nur eine Verbrückung Y pro Molekül enthalten.

Among the binuclear developer components which can be used in the coloring compositions according to the invention, one can name in particular the compounds which correspond to the following formula (II) and their physiologically tolerable salts:


in which:

• - Z ¹ and Z ² independently of one another represent a hydroxyl or NH ₂ radical, the
• optionally substituted by a C ₁ to C ₄ alkyl radical, by a C ₁ to C ₄ hydroxyalkyl radical and / or by a bridging Y,
• the bridge Y represents an alkylene group with 1 to 14 carbon atoms,
• - Such as a linear or branched alkylene chain or an alkylene ring which can be interrupted or terminated by one or more nitrogen-containing groups and / or one or more heteroatoms such as oxygen, sulfur or nitrogen atoms and possibly by one or more hydroxyl or C ₁ until C ₈ -alkoxy radicals can be substituted,
• G ⁵ and G ⁶ independently of one another represent a hydrogen or halogen atom,
• a C ₁ to C ₄ alkyl radical, a C ₁ to C ₄ monohydroxyalkyl radical, a C ₂ to C ₄ polyhydroxyalkyl radical, a C ₁ to C ₄ aminoalkyl radical or a direct compound for bridging Y,
• G ⁷ , G ⁸ , G ⁹ , G ¹⁰ , G ¹¹ and G ¹² independently of one another represent a hydrogen atom, a direct bond to the bridging Y or a C ₁ to C ₄ alkyl radical,
  with the proviso that the compounds of formula (II) contain only one bridge Y per molecule.

According to the invention, the substituents used in formula (II) are analogous to defined above.

Preferred dinuclear developer components of the formula (II) are in particular: N, N'-bis (β-hydroxyethyl) -N, N'-bis (4'-aminophenyl) -1,3-diaminopropanol, N, N'-bis (β-hydroxyethyl) -N, N ' bis (4'-aminophenyl) ethylenediamine, N, N'-bis (4-aminophenyl) tetramethylene diamine, N, N'-bis (β-hydroxyethyl) -N, N'- bis- (4-aminophenyl) tetramethylene diamine, N, N'-bis (4-methylaminophenyl) - tetramethylene diamine, N, N'-bis (ethyl) -N, N'-bis (4'-amino-3'-methylphenyl) - ethylenediamine, 1,8-bis (2,5-diaminophenoxy) -3,5-dioxaoctane, bis- (2-hydroxy-5- aminophenyl) methane, 1,4-bis (4-aminophenyl) diazacycloheptane and 1.10- Bis- (2,5-diaminophenyl) -1,4,7,10-tetraoxadecane and its physiological tolerable salts.

Very particularly preferred binuclear developer components of the formula (II) are N, N'-bis- (β-hydroxyethyl) -N, N'-bis- (4'-aminophenyl) -1,3-diaminopropanol, bis- (2-hydroxy-5-aminophenyl) methane, N, N '-bis- (4-aminophenyl) - 1,4-diazacycloheptane and 1,10-bis (2,5-diaminophenyl) -1,4,7,10-tetraoxadecane or one of their physiologically acceptable salts.

• - G¹³ steht für ein Wasserstoffatom, ein Halogenatom, ein C₁- bis C₄- Alkylradikal, ein C₁- bis C₄-Monohydroxyalkylradikal, ein (C₁- bis C₄)-Alkoxy- (C₁- bis C₄)-alkylradikal, ein C₁- bis C₄-Aminoalkylradikal, ein Hydroxy-(C₁- bis C₄)-alkylaminoradikal, ein C₁- bis C₄-Hydroxyalkoxyradikal, ein C₁- bis C₄-Hydroxyalkyl-(C₁- bis C₄)-aminoalkylradikal oder ein (Di-C₁- bis C₄- Alkylamino)-(C₁- bis C₄)-alkylradikal, und
• - G¹⁴ steht für ein Wasserstoff- oder Halogenatom, ein C₁- bis C₄- Alkylradikal, ein C₁- bis C₄-Monohydroxyalkylradikal, ein C₂- bis C₄- Polyhydroxyalkylradikal, ein (C₁- bis C₄)-Alkoxy-(C₁- bis C₄)-alkylradikal, ein C₁- bis C₄-Aminoalkylradikal oder ein C₁- bis C₄-Cyanoalkylradikal,
• - G¹⁵ steht für Wasserstoff, ein C₁- bis C₄-Alkylradikal, ein C₁- bis C₄- Monohydroxyalkylradikal, ein C₂- bis C₄-Polyhydroxyalkylradikal, ein Phenylradikal oder ein Benzylradikal, und
• - G¹⁶ steht für Wasserstoff oder ein Halogenatom.

Furthermore, it can be preferred according to the invention to use a p-aminophenol derivative or one of its physiologically tolerable salts as developer component. P-Aminophenol derivatives of the formula (III) are particularly preferred


in which:

• - G ¹³ represents a hydrogen atom, a halogen atom, a C ₁ to C ₄ alkyl radical, a C ₁ to C ₄ monohydroxyalkyl radical, a (C ₁ to C ₄ ) alkoxy (C ₁ to C ₄ ) alkyl radical, a C ₁ - to C ₄ -aminoalkyl radical, a hydroxy (C ₁ - to C -alkylaminoradikal _4), a C ₁ - to C ₄ -Hydroxyalkoxyradikal, a C ₁ - to C ₄ hydroxyalkyl (C ₁ - to C ₄ ) -aminoalkyl radical or a (di-C ₁ to C ₄ -alkylamino) - (C ₁ - to C ₄ ) -alkyl radical, and
• G ¹⁴ represents a hydrogen or halogen atom, a C ₁ to C ₄ alkyl radical, a C ₁ to C ₄ monohydroxyalkyl radical, a C ₂ to C ₄ polyhydroxyalkyl radical, a (C ₁ to C ₄ ) Alkoxy- (C ₁ - to C ₄ ) -alkyl radical, a C ₁ - to C ₄ -aminoalkyl radical or a C ₁ - to C ₄ -cyanoalkyl radical,
• G ¹⁵ represents hydrogen, a C ₁ to C ₄ alkyl radical, a C ₁ to C ₄ monohydroxyalkyl radical, a C ₂ to C ₄ polyhydroxyalkyl radical, a phenyl radical or a benzyl radical, and
• - G ¹⁶ represents hydrogen or a halogen atom.

According to the invention, the substituents used in formula (III) are analogous to defined above.

Preferred p-aminophenols of the formula (III) are in particular p-aminophenol, N-methyl-p-aminophenol, 4-amino-3-methylphenol, 4-amino-3-fluorophenol, 2- Hydroxymethylamino-4-aminophenol, 4-amino-3-hydroxymethylphenol, 4- Amino-2- (2-hydroxyethoxy) phenol, 4-amino-2-methylphenol, 4-amino-2- hydroxymethylphenol, 4-amino-2-methoxymethyl-phenol, 4-amino-2- aminomethylphenol, 4-amino-2- (β-hydroxyethyl-aminomethyl) phenol, 4-amino- 2-fluorophenol, 4-amino-2-chlorophenol, 2,6-dichloro-4-aminophenol, 4-amino-2- ((diethylamino) methyl) phenol and their physiologically acceptable salts.

Very particularly preferred compounds of the formula (III) are p-aminophenol, 4-amino-3-methylphenol, 4-amino-2-chlorophenol, 4-amino-2,6-dichlorophenol, 4- Amino-2-aminomethylphenol and 4-amino-2 - ((diethylamino) methyl) phenol.

Furthermore, the developer component can be selected from o-aminophenol and its derivatives, such as 2-amino-4-methylphenol or 2- Amino-4-chlorophenol.

Furthermore, the developer component can be selected from heterocyclic developer components, such as the pyridine, pyrimidine, Pyrazole, pyrazole-pyrimidine derivatives and their physiologically compatible Salt.

Preferred pyridine derivatives are in particular the compounds that are in the patents GB 1 026 978 and GB 1 153 196 can be described, such as 2,5-diamino-pyridine, 2- (4-methoxyphenyl) amino-3-aminopyridine, 2,3-diamino-6-methoxypyridine, 2- (β-methoxyethyl) amino-3-amino-6-methoxy-pyridine and 3,4-diamino-pyridine.

Preferred pyrimidine derivatives are in particular the compounds which are in the German patent DE 23 59 399, Japanese laid-open publication JP-A2- 02/019576 or in the published patent application WO 96/15765, such as 2,4,5,6-tetraaminopyrimidine, 4-hydroxy-2,5,6-triaminopyrimidine, 2-hydroxy 4,5,6-triaminopyrimidine, 2-dimethylamino-4,5,6-triaminopyrimidine, 2,4- Dihydroxy-5,6-diaminopyrimidine and 2,5,6-triaminopyrimidine.

Preferred pyrazole derivatives are in particular the compounds described in the patents
DE 38 43 892, DE 41 33 957 and published documents WO 94/08969, WO 94/08970,
EP 0 740 931 and DE 195 43 988 can be described, such as 4,5-diamino-1-methylpyrazole, 4,5-diamino-1- (β-hydroxyethyl) pyrazole, 3,4-diaminopyrazole, 4,5-diamino -1- (4'-chlorobenzyl) pyrazole, 4,5-diamino-1,3-dimethylpyrazole, 4,5-diamino-3-methyl-1-phenylpyrazole, 4,5-diamino-1-methyl-3- phenylpyrazole, 4-amino-1,3-dimethyl-5-hydrazinopyrazole, 1-benzyl-4,5-diamino-3-methylpyrazole, 4,5-diamino-3-tert-butyl-1-methylpyrazole, 4.5 -Diamino-1-tert-butyl-3-methylpyrazole, 4,5-diamino-1- (β-hydroxyethyl) -3-methylpyrazole, 4,5-diamino-1-ethyl-3-methylpyrazole, 4,5- Diamino-1-ethyl-3- (4'-methoxyphenyl) pyrazole, 4,5-diamino-1-ethyl-3-hydroxymethylpyrazole, 4,5-diamino-3-hydroxymethyl-1-methylpyrazole, 4,5-diamino -3-hydroxymethyl-1-isopropylpyrazole, 4,5-diamino-3-methyl-1-isopropylpyrazole, 4-amino-5- (2'-aminoethyl) amino-1,3-dimethylpyrazole, 3,4,5-triaminopyrazole , 1-methyl-3,4,5-triaminopyrazole, 3,5-diamino-1-methyl-4-methylaminopyrazole and 3,5-diamino-4 (β-hydroxyethyl) amino-1-methylpyrazole.

• - G¹⁷, G¹⁸, G¹⁹ und G²⁰ unabhängig voneinander stehen für ein Wasserstoffatom, ein C₁- bis C₄-Alkylradikal, ein Aryl-Radikal, ein C₁- bis C₄- Hydroxyalkylradikal, ein C₂- bis C₄-Polyhydroxyalkylradikal, ein (C₁- bis C₄)-Alkoxy-(C₁- bis C₄)-alkylradikal, ein C₁- bis C₄-Aminoalkylradikal, das gegebenenfalls durch ein Acetyl-Ureid- oder Sulfonyl-Radikal geschützt sein kann, ein (C₁- bis C₄)-Alkylamino-(C₁- bis C₄)-alkylradikal, ein Di- [(C₁- bis C₄)-alkyl]-(C₁- bis C₄)-aminoalkylradikal, wobei die Dialkyl- Radikale gegebenenfalls einen Kohlenstoffzyklus oder einen Heterozyklus mit 5 oder 6 Kettengliedern bilden, ein C₁- bis C₄-Hydroxyalkyl- oder ein Di-(C₁- bis C₄)-[Hydroxyalkyl]-(C₁- bis C₄)-aminoalkylradikal,
• - die X-Radikale stehen unabhängig voneinander für ein Wasserstoffatom, ein C₁- bis C₄-Alkylradikal, ein Aryl-Radikal, ein C₁- bis C₄- Hydroxyalkyladikal, ein C₂- bis C₄- Polyhydroxyalkylradikal, ein C₁- bis C₄-Aminoalkylradikal, ein (C₁- bis C₄)-Alkylamino-(C₁- bis C₄)-alkylradikal, ein Di-[(C₁- bis C₄)alkyl]-(C₁- bis C₄)-aminoalkylradikal, wobei die Dialkyl-Radikale gegebenenfalls einen Kohlenstoffzyklus oder einen Heterozyklus mit 5 oder 6 Kettengliedern bilden, ein C₁- bis C₄-Hydroxyalkyl- oder ein Di-(C₁- bis C₄-hydroxyalkyl)-aminoalkylradikal, ein Aminoradikal, ein C₁- bis C₄-Alkyl- oder Di-(C₁- bis C₄-hydroxyalkyl)-aminoradikal, ein Halogenatom, eine Carboxylsäuregruppe oder eine Sulfonsäuregruppe,
• - i hat den Wert 0, 1, 2 oder 3,
• - p hat den Wert 0 oder 1,
• - q hat den Wert 0 oder 1 und
• - n hat den Wert 0 oder 1,

mit der Maßgabe, dass

• - die Summe aus p + q ungleich 0 ist,
• - wenn p + q gleich 2 ist, n den Wert 0 hat, und die Gruppen N¹⁷G¹⁸ und NG¹⁹G²⁰ belegen die Positionen (2, 3); (5, 6); (6, 7); (3, 5) oder (3, 7);
• - wenn p + q gleich 1 ist, n den Wert 1 hat, und die Gruppen NG¹⁷G¹⁸ (oder NG¹⁹G²⁰) und die Gruppe OH belegen die Positionen (2, 3); (5, 6); (6, 7); (3, 5) oder (3, 7);

Preferred pyrazole-pyrimidine derivatives are, in particular, the derivatives of pyrazole- [1,5-a] pyrimidine of the following formula (IV) and its tautomeric forms, provided that there is a tautomeric equilibrium:


in which:

• G ¹⁷ , G ¹⁸ , G ¹⁹ and G ²⁰ independently of one another represent a hydrogen atom, a C ₁ to C ₄ alkyl radical, an aryl radical, a C ₁ to C ₄ hydroxyalkyl radical, a C ₂ to C ₄ -Polyhydroxyalkyl radical, a (C ₁ - to C ₄ ) -alkoxy- (C ₁ - to C ₄ ) -alkyl radical, a C ₁ - to C ₄ -aminoalkyl radical which is optionally protected by an acetyl-ureide or sulfonyl radical can be a (C ₁ - to C ₄ ) -alkylamino- (C ₁ - to C ₄ ) -alkyl radical, a di- [(C ₁ - to C ₄ ) -alkyl] - (C ₁ - to C ₄ ) -aminoalkyl radical, where the dialkyl radicals optionally form a carbon cycle or a heterocycle with 5 or 6 chain links, a C ₁ - to C ₄ -hydroxyalkyl or a di- (C ₁ - to C ₄ ) - [hydroxyalkyl] - (C ₁ - to C ₄ ) -aminoalkyl radical,
• - The X radicals independently of one another represent a hydrogen atom, a C ₁ to C ₄ alkyl radical, an aryl radical, a C ₁ to C ₄ hydroxyalkyl radical, a C ₂ to C ₄ polyhydroxyalkyl radical, a C ₁ - to C ₄ -aminoalkyl radical, a (C ₁ - to C ₄ ) -alkylamino- (C ₁ - to C ₄ ) -alkyl radical, a di - [(C ₁ - to C ₄ ) alkyl] - (C ₁ - bis C ₄ ) aminoalkyl radical, where the dialkyl radicals optionally form a carbon cycle or a heterocycle with 5 or 6 chain links, a C ₁ - to C ₄ -hydroxyalkyl or a di- (C ₁ - to C ₄ -hydroxyalkyl) aminoalkyl radical , an amino radical, a C ₁ to C ₄ alkyl or di (C ₁ to C ₄ hydroxyalkyl) amino radical, a halogen atom, a carboxylic acid group or a sulfonic acid group,
• - i has the value 0, 1, 2 or 3,
• - p has the value 0 or 1,
• - q has the value 0 or 1 and
• - n has the value 0 or 1,

with the proviso that

• - the sum of p + q is not equal to 0,
• - if p + q is 2, n has the value 0, and the groups N ¹⁷ G ¹⁸ and NG ¹⁹ G ²⁰ occupy positions (2, 3); (5, 6); (6, 7); (3, 5) or (3, 7);
• - if p + q is 1, n has the value 1, and the groups NG ¹⁷ G ¹⁸ (or NG ¹⁹ G ²⁰ ) and the group OH occupy positions (2, 3); (5, 6); (6, 7); (3, 5) or (3, 7);

According to the invention, the substituents used in formula (IV) are analogous to defined above.

If the pyrazole- [1,5-a] pyrimidine of the formula (IV) above contains a hydroxy group at one of the positions 2, 5 or 7 of the ring system, there is a tautomeric equilibrium, which is illustrated in the following scheme, for example:

• - Pyrazol-[1,5-a]-pyrimidin-3,7-diamin;
• - 2,5-Dimethyl pyrazol-[1,5-a]-pyrimidin-3,7-diamin;
• - Pyrazol-(1,5-a]-pyrimidin-3,5-diamin;
• - 2,7-Dimethyl pyrazol-[1,5-a]-pyrimidin-3,5-diamin;
• - 3-Amino pyrazol-[1,5-a]-pyrimidin-7-ol;
• - 3-Amino pyrazol-[1,5-a]-pyrimidin-5-ol;
• - 2-(3-Amino pyrazol-[1,5-a]-pyrimidin-7-ylamino)-ethanol;
• - 2-(7-Amino pyrazol-[1,5-a]-pyrimidin-3-ylamino)-ethanol;
• - 2-[(3-Amino pyrazol-[1,5-a]-pyrimidin-7-yl)-(2-hydroxy-ethyl)-amino]- ethanol;
• - 2-[(7-Amino pyrazol-[1,5-a]-pyrimidin-3-yl)-(2-hydroxy-ethyl)-amino]- ethanol;
• - 5,6-Dimethyl pyrazol-[1,5-a]-pyrimidin-3,7-diamin;
• - 2,6-Dimethyl pyrazol-[1,5-a]-pyrimidin-3,7-diamin;
• - 2,5,N7,N7-Tetramethyl pyrazol-[1,5-a]-pyrimidin-3,7-diamin;

sowie ihre physiologisch verträglichen Salze und ihre tautomeren Formen, wenn ein tautomerisches Gleichgewicht vorhanden ist. Among the pyrazole- [1,5-a] pyrimidines of the above formula (IV) one can mention in particular:

• - pyrazole- [1,5-a] pyrimidine-3,7-diamine;
• - 2,5-dimethyl pyrazole- [1,5-a] pyrimidine-3,7-diamine;
• - pyrazole- (1,5-a] pyrimidine-3,5-diamine;
• - 2,7-dimethyl pyrazole- [1,5-a] pyrimidine-3,5-diamine;
• - 3-amino pyrazol- [1,5-a] pyrimidin-7-ol;
• - 3-amino pyrazol- [1,5-a] pyrimidin-5-ol;
• - 2- (3-amino pyrazole- [1,5-a] pyrimidin-7-ylamino) ethanol;
• - 2- (7-amino pyrazole- [1,5-a] pyrimidin-3-ylamino) ethanol;
• - 2 - [(3-amino pyrazol- [1,5-a] pyrimidin-7-yl) - (2-hydroxyethyl) amino] ethanol;
• - 2 - [(7-amino pyrazol- [1,5-a] pyrimidin-3-yl) - (2-hydroxyethyl) amino] ethanol;
• - 5,6-dimethyl pyrazole- [1,5-a] pyrimidine-3,7-diamine;
• - 2,6-dimethyl pyrazole- [1,5-a] pyrimidine-3,7-diamine;
• - 2,5, N7, N7-tetramethyl pyrazole- [1,5-a] pyrimidine-3,7-diamine;

as well as their physiologically acceptable salts and their tautomeric forms when a tautomeric equilibrium is present.

The pyrazole [1,5-a] pyrimidines of the above formula (IV) can be as in described in the literature by cyclization starting from one Aminopyrazole or be made by hydrazine.

Usually m-phenylenediamine derivatives, Naphthols, resorcinol and resorcinol derivatives, pyrazolones and m-Aminophenol derivatives used. 1- are particularly suitable as coupler substances Naphthol, 1,5-, 2,7- and 1,7-dihydroxynaphthalene, 5-amino-2-methylphenol, m- Aminophenol, resorcinol, resorcinol monomethyl ether, m-phenylenediamine, 1-phenyl-3-methyl-pyrazolon-5, 2,4-dichloro-3-aminophenol, 1,3-bis (2,4-diaminophenoxy) propane, 2-chloro-resorcinol, 4-chloro-resorcinol, 2-chloro-6-methyl-3-aminophenol, 2-amino-3-hydroxypyridine, 2-methylresorcinol, 5-methylresorcinol and 2- Methyl 4-chloro-5-aminophenol.

• - m-Aminophenol und dessen Derivate wie beispielsweise 5-Amino-2- methylphenol, 3-Amino-2-chlor-6-methylphenol, 2-Hydroxy-4- aminophenoxyethanol, 2,6-Dimethyl-3-aminophenol, 3-Trifluoroacetylamino- 2-chlor-6-methylphenol, 5-Amino-4-chlor-2-methylphenol, 5-Amino-4- methoxy-2-methylphenol, 5-(2'-Hydroxyethyl)-amino-2-methylphenol, 3- (Diethylamino)-phenol, N-Cyclopentyl-3-aminophenol, 1,3-Dihydroxy-5- (methylamino)-benzol, 3-(Ethylamino)-4-methylphenol und 2,4-Dichlor-3- aminophenol,
• - o-Aminophenol und dessen Derivate,
• - m-Diaminobenzol und dessen Derivate wie beispielsweise 2,4- Diaminophenoxyethanol, 1,3-Bis-(2,4-diaminophenoxy)-propan, 1-Methoxy- 2-amino-4-(2'-hydroxyethylamino)benzol, 1,3-Bis-(2,4-diaminophenyl)- propan, 2,6-Bis-(2-hydroxyethylamino)-1-methylbenzol und 1-Amino-3-bis- (2'-hydroxyethyl)-aminobenzol,
• - o-Diaminobenzol und dessen Derivate wie beispielsweise 3,4- Diaminobenzoesäure und 2,3-Diamino-1-methylbenzol,
• - Di- beziehungsweise Trihydroxybenzolderivate wie beispielsweise Resorcin, Resorcinmonomethylether, 2-Methylresorcin, 5-Methylresorcin, 2,5- Dimethylresorcin, 2-Chlorresorcin, 4-Chlorresorcin, Pyrogallol und 1,2,4- Trihydroxybenzol,
• - Pyridinderivate wie beispielsweise 2,6-Dihydroxypyridin, 2-Amino-3- hydroxypyridin, 2-Amino-5-chlor-3-hydroxypyridin, 3-Amino-2-methylamino- 6-methoxypyridin, 2,6-Dihydroxy-3,4-dimethylpyridin, 2,6-Dihydroxy-4- methylpyridin, 2,6-Diaminopyridin, 2,3-Diamino-6-methoxypyridin und 3,5- Diamino-2,6-dimethoxypyridin,
• - Naphthalinderivate wie beispielsweise 1-Naphthol, 2-Methyl-1-naphthol, 2- Hydroxymethyl-1-naphthol, 2-Hydroxyethyl-1-naphthol, 1,5- Dihydroxynaphthalin, 1,6-Dihydroxynaphthalin, 1,7-Dihydroxynaphthalin, 1,8-Dihydroxynaphthalin, 2,7-Dihydroxynaphthalin und 2,3- Dihydroxynaphthalin,
• - Morpholinderivate wie beispielsweise 6-Hydroxybenzomorpholin und 6- Amino-benzomorpholin,
• - Chinoxalinderivate wie beispielsweise 6-Methyl-1,2,3,4-tetrahydrochinoxalin,
• - Pyrazolderivate wie beispielsweise 1-Phenyl-3-methylpyrazol-5-on,
• - Indolderivate wie beispielsweise 4-Hydroxyindol, 6-Hydroxyindol und 7- Hydroxyindol,
• - Pyrimidinderivate, wie beispielsweise 4,6-Diaminopyrimidin, 4-Amino-2,6- dihydroxypyrimidin, 2,4-Diamino-6-hydroxypyrimidin, 2,4,6- Trihydroxypyrimidin, 2-Amino-4-methylpyrimidin, 2-Amino-4-hydroxy-6- methylpyrimidin und 4,6-Dihydroxy-2-methylpyrimidin, oder
• - Methylendioxybenzolderivate wie beispielsweise 1-Hydroxy-3,4- methylendioxybenzol, 1-Amino-3,4-methylendioxybenzol und 1-(2'- Hydroxyethyl)-amino-3,4-methylendioxybenzol,

Coupler components preferred according to the invention are:

• - m-Aminophenol and its derivatives such as 5-amino-2-methylphenol, 3-amino-2-chloro-6-methylphenol, 2-hydroxy-4-aminophenoxyethanol, 2,6-dimethyl-3-aminophenol, 3-trifluoroacetylamino - 2-chloro-6-methylphenol, 5-amino-4-chloro-2-methylphenol, 5-amino-4-methoxy-2-methylphenol, 5- (2'-hydroxyethyl) amino-2-methylphenol, 3- (Diethylamino) phenol, N-cyclopentyl-3-aminophenol, 1,3-dihydroxy-5- (methylamino) benzene, 3- (ethylamino) -4-methylphenol and 2,4-dichloro-3-aminophenol,
• o-aminophenol and its derivatives,
• m-diamino benzene and its derivatives such as, for example, 2,4-diaminophenoxyethanol, 1,3-bis (2,4-diaminophenoxy) propane, 1-methoxy-2-amino-4- (2'-hydroxyethylamino) benzene, 1 , 3-bis (2,4-diaminophenyl) propane, 2,6-bis (2-hydroxyethylamino) -1-methylbenzene and 1-amino-3-bis (2'-hydroxyethyl) aminobenzene,
• o-diamino benzene and its derivatives such as 3,4-diamino benzoic acid and 2,3-diamino-1-methylbenzene,
• - Di- or trihydroxybenzene derivatives such as resorcinol, resorcinol monomethyl ether, 2-methylresorcinol, 5-methylresorcinol, 2,5-dimethylresorcinol, 2-chlororesorcinol, 4-chlororesorcinol, pyrogallol and 1,2,4-trihydroxybenzene,
• Pyridine derivatives such as 2,6-dihydroxypyridine, 2-amino-3-hydroxypyridine, 2-amino-5-chloro-3-hydroxypyridine, 3-amino-2-methylamino-6-methoxypyridine, 2,6-dihydroxy-3, 4-dimethylpyridine, 2,6-dihydroxy-4-methylpyridine, 2,6-diaminopyridine, 2,3-diamino-6-methoxypyridine and 3,5-diamino-2,6-dimethoxypyridine,
• Naphthalene derivatives such as 1-naphthol, 2-methyl-1-naphthol, 2-hydroxymethyl-1-naphthol, 2-hydroxyethyl-1-naphthol, 1,5-dihydroxynaphthalene, 1,6-dihydroxynaphthalene, 1,7-dihydroxynaphthalene, 1,8-dihydroxynaphthalene, 2,7-dihydroxynaphthalene and 2,3-dihydroxynaphthalene,
• Morpholine derivatives such as 6-hydroxybenzomorpholine and 6-amino-benzomorpholine,
• Quinoxaline derivatives such as 6-methyl-1,2,3,4-tetrahydroquinoxaline,
• Pyrazole derivatives such as 1-phenyl-3-methylpyrazol-5-one,
• Indole derivatives such as 4-hydroxyindole, 6-hydroxyindole and 7-hydroxyindole,
• - Pyrimidine derivatives, such as 4,6-diaminopyrimidine, 4-amino-2,6-dihydroxypyrimidine, 2,4-diamino-6-hydroxypyrimidine, 2,4,6-trihydroxypyrimidine, 2-amino-4-methylpyrimidine, 2-amino -4-hydroxy-6-methylpyrimidine and 4,6-dihydroxy-2-methylpyrimidine, or
• Methylenedioxybenzene derivatives such as 1-hydroxy-3,4-methylenedioxybenzene, 1-amino-3,4-methylenedioxybenzene and 1- (2'-hydroxyethyl) amino-3,4-methylenedioxybenzene,

Particularly preferred coupler components are 1-naphthol, 1,5-, 2,7- and 1,7-dihydroxynaphthalene, 3-aminophenol, 5-amino-2-methylphenol, 2-amino-3- hydroxypyridine, resorcinol, 4-chlororesorcinol, 2-chloro-6-methyl-3-aminophenol, 2- Methylresorcinol, 5-methylresorcinol, 2,5-dimethylresorcinol and 2,6-dihydroxy-3,4- dimethylpyridine.

It is not necessary for the oxidation dye precursors or the direct dyes each represent uniform compounds. Much more can in the hair dye according to the invention, due to the Manufacturing process for the individual dyes, in minor quantities other components may be included, unless they are Adversely affect the coloring result or for other reasons, e.g. B. toxicological, must be excluded.

Regarding the hair colorants and tints of the invention usable dyes will continue to expressly on the monograph Ch. Zviak, The Science of Hair Care, Chapter 7 (pages 248-250; substantive Dyes) and Chapter 8, pages 264-267; Oxidation dye precursors) published as volume 7 of the series "Dermatology" (ed .: Ch., Culnan and H. Maibach), publisher Marcel Dekker Inc., New York, Basel, 1986, and the "European inventory of cosmetic raw materials", published by the European Community, available in disk form from the Federal Association of German Industrial and trading company for pharmaceuticals, health products and Personal care products e. V., Mannheim, referred to.

The oxidation dye precursors are in the agents according to the invention preferably in amounts of 0.01 to 20% by weight, preferably 0.5 to 5% by weight, in each case based on the total composition.

Such indoles and. Are preferred as precursors of nature-analogous dyes Indolines used which have at least one hydroxyl or amino group, preferably as a substituent on the six-membered ring. These groups can do more Wear substituents, e.g. B. in the form of etherification or esterification of Hydroxy group or an alkylation of the amino group.

• - R¹ steht für Wasserstoff, eine C₁-C₄-Alkylgruppe oder eine C₁-C₄ -Hydroxyalkylgruppe,
• - R² steht für Wasserstoff oder eine -COOH-Gruppe, wobei die -COOH- Gruppe auch als Salz mit einem physiologisch verträglichen Kation vorliegen kann,
• - R³ steht für Wasserstoff oder eine C₁-C₄-Alkylgruppe,
• - R⁴ steht für Wasserstoff, eine C₁-C₄-Alkylgruppe oder eine Gruppe -CO-R⁶, in der R⁶ steht für eine C₁-C₄-Alkylgruppe, und
• - R⁵ steht für eine der unter R⁴ genannten Gruppen,

sowie physiologisch verträgliche Salze dieser Verbindungen mit einer organischen oder anorganischen Säure. Derivatives of 5,6-dihydroxyindoline of the formula (Va) are particularly suitable as precursors of naturally analogous hair dyes,


in the independently of each other

• R ¹ represents hydrogen, a C ₁ -C ₄ -alkyl group or a C ₁ -C ₄ -hydroxyalkyl group,
• R ² stands for hydrogen or a -COOH group, where the -COOH group can also be present as a salt with a physiologically compatible cation,
• R ³ represents hydrogen or a C ₁ -C ₄ alkyl group,
• - R ⁴ stands for hydrogen, a C ₁ -C ₄ alkyl group or a group -CO-R ⁶ , in which R ⁶ stands for a C ₁ -C ₄ alkyl group, and
• R ⁵ stands for one of the groups mentioned under R ⁴ ,

as well as physiologically tolerable salts of these compounds with an organic or inorganic acid.

Particularly preferred derivatives of indoline are 5,6-dihydroxyindoline, N-methyl-5,6-dihydroxyindoline, N-ethyl-5,6-dihydroxyindoline, N-propyl-5,6-dihydroxyindoline,
N-butyl-5,6-dihydroxyindoline, 5,6-dihydroxyindoline-2-carboxylic acid and the 6-hydroxyindoline, the 6-aminoindoline and the 4-aminoindoline.

Of particular note within this group are N-methyl-5,6- dihydroxyindoline, N-ethyl-5,6-dihydroxyindoline, N-propyl-5,6-dihydroxyindoline, N-butyl-5,6-dihydroxyindoline and especially 5,6-dihydroxyindoline.

• - R¹ steht für Wasserstoff, eine C₁-C₄-Alkylgruppe oder eine C₁-C₄ -Hydroxyalkylgruppe,
• - R² steht für Wasserstoff oder eine -COOH-Gruppe, wobei die -COOH- Gruppe auch als Salz mit einem physiologisch verträglichen Kation vorliegen kann,
• - R³ steht für Wasserstoff oder eine C₁-C₄-Alkylgruppe,
• - R⁴ steht für Wasserstoff, eine C₁-C₄-Alkylgruppe oder eine Gruppe -CO-R⁶, in der R⁶ steht für eine C₁-C₄-Alkylgruppe, und
• - R⁵ steht für eine der unter R⁴ genannten Gruppen,
• - sowie physiologisch verträgliche Salze dieser Verbindungen mit einer organischen oder anorganischen Säure.

Derivatives of 5,6-dihydroxyindole of the formula (Vb) are also outstandingly suitable as precursors of natural hair dyes,


in the independently of each other

• R ¹ represents hydrogen, a C ₁ -C ₄ -alkyl group or a C ₁ -C ₄ -hydroxyalkyl group,
• R ² stands for hydrogen or a -COOH group, where the -COOH group can also be present as a salt with a physiologically compatible cation,
• R ³ represents hydrogen or a C ₁ -C ₄ alkyl group,
• - R ⁴ stands for hydrogen, a C ₁ -C ₄ alkyl group or a group -CO-R ⁶ , in which R ⁶ stands for a C ₁ -C ₄ alkyl group, and
• R ⁵ stands for one of the groups mentioned under R ⁴ ,
• - And physiologically acceptable salts of these compounds with an organic or inorganic acid.

Particularly preferred derivatives of indole are 5,6-dihydroxyindole, N-methyl 5,6-dihydroxyindole, N-ethyl-5,6-dihydroxyindole, N-propyl-5,6-dihydroxyindole, N- Butyl-5,6-dihydroxyindole, 5,6-dihydroxyindole-2-carboxylic acid, 6-hydroxyindole, 6- Aminoindole and 4-aminoindole.

Of particular note within this group are N-methyl-5,6-dihydroxyindole, N- Ethyl-5,6-dihydroxyindole, N-propyl-5,6-dihydroxyindole, N-butyl-5,6- dihydroxyindole and in particular 5,6-dihydroxyindole.

The indoline and indole derivatives can be used in the Colorants used according to the method both as free bases and in Form their physiologically compatible salts with inorganic or organic acids, e.g. B. the hydrochloride, sulfate and hydrobromide, be used. The indole or indoline derivatives are usually in these Contain amounts of 0.05-10 wt .-%, preferably 0.2-5 wt .-%.

In a further embodiment, it can be preferred according to the invention the indoline or indole derivative in hair dyes in combination with use at least one amino acid or an oligopeptide. The amino acid is is advantageously an α-amino acid; very particularly preferred α- Amino acids are arginine, ornithine, lysine and histidine, especially arginine.

In addition to the dye precursors, the colorants according to the invention contain direct dyes for further shading. These are usually selected from nitrophenylenediamines, nitroaminophenols, Azo dyes, anthraquinones or indophenols. Preferred direct Dyes are those under the international names or trade names HC Yellow 2, HC Yellow 4, HC Yellow 5, HC Yellow 6, Basic Yellow 57, HC Orange 1, Disperse Orange 3, HC Red 1, HC Red 3, HC Red 13, HC Red BN, Basic Red 76, HC Blue 2, HC Blue 12, Disperse Blue 3, Basic Blue 7, Basic Blue 26, Basic Blue 99, HC Violet 1, Disperse Violet 1, Disperse Violet 4, Basic Violet 2, Basic Violet 14, Acid Violet 43, Disperse Black 9, Acid Black 52, Basic Brown 16 and Basic Brown 17 known compounds as well as 1,4-bis- (β- hydroxyethyl) amino-2-nitrobenzene, 3-nitro-4- (β-hydroxyethyl) aminophenol, 4- Amino-2-nitrodiphenylamine-2'-carboxylic acid, 6-nitro-1,2,3,4-tetrahydroquinoxaline, 2-hydroxy-1,4-naphthoquinone, hydroxyethyl-2-nitro-toluidine, picramic acid, 2- Amino-6-chloro-4-nitrophenol, 4-ethylamino-3-nitrobenzoic acid and 2-chloro- 6-ethylamino-1-hydroxy-4-nitrobenzene. The agents according to the invention In this embodiment, the substantive dyes preferably contain an amount of 0.01 to 20 wt .-%, based on the total colorant.

Furthermore, the preparations according to the invention can also be used in nature occurring dyes such as henna red, henna neutral, henna black, chamomile flower, sandalwood, black tea, sapwood, sage, Contains blue wood, madder root, catechu, sedre and alkanna root.

In addition to the enzyme system obtained according to the invention, the Colorants of the invention also other enzymes, such as Contain laccases, peroxidases or oxidases with their respective substrates. Preferred oxidases are, for example, choline oxidase, glucose oxidase, Alcohol oxidase, pyruvate oxidase, oxalate oxidase, cholesterol oxidase, Uricase, lactate oxidase, xanthine oxidase, pyranose oxidase, glycerin oxidase and galactose oxidase. Especially in this embodiment preferred are agents which, in addition to the phenol oxidizing enzyme Uricase, glucose oxidase and / or xanthine oxidase and their respective Contain substrates.

To produce the colorants according to the invention, the Dye precursors are incorporated into a suitable water-containing carrier. For the purpose of hair coloring, such carriers are e.g. B. creams, emulsions, Gels or surfactant-containing foaming solutions, e.g. B. shampoos, Foam aerosols or other preparations intended for use on the hair are suitable.

The colorants according to the invention can all be used for such Contain preparations known active ingredients, additives and auxiliaries. In many cases the colorants contain at least one surfactant, in principle both anionic as well as zwitterionic, ampholytic, nonionic and cationic Surfactants are suitable. A person skilled in the art can determine the possible influence of the various Surfactants on the activity of the enzyme system according to the invention Check if necessary with simple preliminary tests.

In a preferred embodiment of the present invention in the Agents for coloring keratin fibers a combination of anionic and nonionic surfactants or a combination of anionic and amphoteric surfactants used.

In individual cases, however, it has proven advantageous to use the surfactants choose amphoteric or non-ionic surfactants, as these usually influence the dyeing process according to the invention less.

pictures

Fig. 1 shows the course of the oxygen transfer rate (OTR) for S. bisbyi in Stachybotrys minimal medium (SMM) according to the invention and in minimal medium according to the KOROLJOVA protocol with the addition of 1 mL / L benzyl alcohol.

Fig. 2 shows the corresponding to Fig. 1, curves of the Phenoloxidaseaktivitäten for S. bisbyi in SMM (0 and 0.025 g / L copper sulfate), and in minimal medium according KOROLJOVA protocol with the addition of 1 ml / L benzyl alcohol.

Fig. 3 shows the comparison of phenol oxidase production in SMM with varied benzyl alcohol additives (0 ml / L, 1 ml / L, 5 ml / L).

The following examples illustrate the invention without, however, pointing to it limit:

1. Production of phenol oxidase by Stachybotrys bisbyi

Stachybotrys bisbyi was used as the enzyme generator.

• a) Die CuSO₄.5H₂O - Konzentration beträgt 0,025 g pro Liter Medium (0,25 g/L nach KOROLJOVA-SKOROBOGAT'KO et al.),
• b) Das erfindungsgemäße Stachybotrys Minimalmedium (SMM) enthält 1 mL/L Benzylalkohol (das Medium nach KOROLJOVA- SKOROBOGAT'KO et al. enthält keinen Benzylalkohol) und
• c) Der pH des Mediums beträgt 5,6 (6,0 nach KOROLJOVA- SKOROBOGAT'KO et al.).

The Stachybotrys minimal medium according to the invention had the following composition:


which is different from that developed by KOROLJOVA-SKOROBOGAT'KO et al. (1998) differs as follows:

• a) The CuSO ₄ .5H ₂ O concentration is 0.025 g per liter of medium (0.25 g / L according to KOROLJOVA-SKOROBOGAT'KO et al.),
• b) The Stachybotrys minimal medium (SMM) according to the invention contains 1 ml / L benzyl alcohol (the medium according to KOROLJOVASKOROBOGAT'KO et al. contains no benzyl alcohol) and
• c) The pH of the medium is 5.6 (6.0 according to KOROLJOVASKOROBOGAT'KO et al.).

Fig. 1 shows the course of the oxygen transfer rate (OTR) for S. bisbyi in Stachybotrys minimal medium (SMM) according to the invention and in minimal medium according to the KOROLJOVA protocol with the addition of 1 mL / L benzyl alcohol. The oxygen transfer rate (OTR) describes the amount of oxygen passing from the gas space into the liquid per culture volume and time. This value shows the respiratory activity or the biological activity of a microbial culture at a certain point in time. The cultivation temperature was 26 ° C, the inoculation density 10 ⁴ spores per ml.

Reduction of the copper sulfate concentration

The minimal medium according to the KOROLJOVA protocol contains 0.25 g / L copper sulfate. SMM contains 0.025 g / L copper sulfate.

Another SMM approach was carried out without copper sulfate (0 g / L).

By reducing the copper sulfate concentration in SMM to 0 or 0.025 g / L the growth of Stachybotrys bibsyi was accelerated by 48 hours and the maximum respiratory rate can be increased by about a quarter (e.g. from 0.0015 mol / L / h to 0.002 mol / L / h).

The maximum derived from the course of the oxygen transfer rate specific growth rate in minimal medium according to the Koroljova protocol (0.25 g / L Copper sulfate) approx. 0.05 1 / h. In SMM medium (0 g / L and 0.025 g / L Copper sulfate), it was increased to about 0.1 l / h.

According to KOROLJOVA-SKOROBOGAT'KO et al. (1998) Copper sulfate concentrations of 0.25 g / L appeared to be inhibitory or toxic.

Fig. 2 shows the corresponding to Fig. 1, curves of the Phenoloxidaseaktivitäten for S. bisbyi in SMM (0 and 0.025 g / L copper sulfate), and in minimal medium according KOROLJOVA protocol with the addition of 1 ml / L benzyl alcohol.

The phenol oxidase production in SMM with 0.025 g / L copper sulfate was accelerated by 96 h compared to the minimal medium according to Koroljova and in SMM the maximum activity of 7 U [ΔE ₄₂₀ / min] was measured.

During the same cultivation period, an activity of only 2 U [ΔE ₄₂₀ / min] was measured in minimal medium according to the Koroljova protocol with 1 mL / L benzyl alcohol.

A phenol oxidase activity of only 0.3 U [ΔE ₄₂₀ / min] was measured in SMM medium without copper sulfate.

A small amount of copper sulfate is required for phenol oxidase production apparently essential.

Induction effect of benzyl alcohol

Fig. 3 shows the comparison of phenol oxidase production in SMM with varied benzyl alcohol additives (0 ml / L, 1 ml / L, 5 ml / L).

The maximum activity of 6 U [ΔE ₄₂₀ / min] was measured at 1 ml / L benzyl alcohol under the examined benzyl alcohol concentrations.

Without KOROLJOVA's benzyl alcohol, lower maximum activities of 3 U [ΔE ₄₂₀ / min] were measured.

When using 5 ml / L benzyl alcohol, the growth remained due to a apparently toxic effects of benzyl alcohol completely.

inoculation

Stachybotrys spec. forms conidiospores on potato dextrose agar after incubation at 26 ° C after four weeks. A stock solution of conidiospores was prepared by suspension in a NaCl solution (0.9 g / L). The inoculation was carried out with a titer of 10 ⁴ spores / ml main culture volume.

cultivation temperature

At a cultivation temperature of 26 ° C, consistently reproducible maximum phenol oxidase activities of 9 U [ΔE ₄₂₀ / min] were measured under media-optimized conditions (see above).

At 18 ° C, 22 ° C and 30 ° C the measured maximum activities varied between 0 and 9 U [ΔE ₄₂₀ / min]. At 18 ° C and 22 ° C, reduced growth was observed at the same time.

Harvesting the culture supernatant

The cultivation period was approximately two weeks. The maximum phenol oxidase Activity was detected after 10 ± 2 days. The phenol oxidase was harvested centrifuged the culture broth and the supernatant for phenol oxidase activity analyzed.

2. Analysis of phenol oxidase activity

• - 1,5 ml 120 mM NaOAc (wasserlöslich) (pH auf 5 einstellen, z. B. mit 1 N NaOH)
• - 0,2 ml 4,5 mM ABTS®
• - 0,3 ml Probe (Kulturüberstand bei 12000 rpm 5 min, abzentrifugiert)
• - kurz durchmischen
• - kontinuierliche Messung der optischen Dichte (OD), z. B. in einem Uvikon- Spektrophotometer bei 420 nm über 2 min. (50 scanning rate pro min.) direkt nach Zugabe des Überstandes.

The phenol oxidase activity was analyzed by oxidizing ABTS® (diammonium salt of 2,2'-azino-bis- (3-ethyl-2,3-dihydro-1,3-benzothiazole-6-sulfonic acid)) as chromogen for the enzymat. Analysis. and by measuring the change in extinction at 420 nm in the photometer according to the following protocol:

• - 1.5 ml 120 mM NaOAc (water soluble) (adjust pH to 5, e.g. with 1 N NaOH)
• - 0.2 ml 4.5 mM ABTS®
• 0.3 ml sample (culture supernatant at 12,000 rpm for 5 min, centrifuged off)
• - mix briefly
• - Continuous measurement of the optical density (OD), e.g. B. in a Uvikon spectrophotometer at 420 nm for 2 min. (50 scanning rate per min.) Directly after adding the supernatant.

1 U corresponds to the change in extinction / min by 1 unit (1 OD) at 420 nm.

3. Bioreactor and shaking conditions

• - Rotationsschüttler, Schüttelkreisdurchmesser 50 mm
• - Füllvolumen 30 mL
• - Schüttelfrequenz 150 Upm

The cultivation was carried out in shaken Erlenmeyer flasks (nominal volume 250 mL). The shaking conditions were as follows:

• - Rotary shaker, shaking circle diameter 50 mm
• - filling volume 30 mL
• - Shaking frequency 150 rpm

The chosen shaking conditions guaranteed an unlimited Oxygen supply.

Reproducibility was improved by using hydrophobized flasks the mushroom morphology can be increased. Formed in this flask consistently pellets with a diameter of approx. 1-5 mm.

Using hydrophobic flasks became stronger maximums Breathing rates and faster growth than observed in untreated flasks. Furthermore, a reproducible one could also be used with hydrophobic flasks maximum phenol oxidase formation can be guaranteed.

In order to check the influence of the glass surface on the culture behavior of the Stachybotrys strains, the glass inner wall of the shake flasks was made hydrophobic. The procedure for the hydrophobization was as follows:
To activate the glass for hydrophobization, the flasks must be hydrophilized beforehand.

a) Instructions for hydrophilizing

The pistons become two thirds of their nominal volume with 20% (v / v) Nitric acid filled and placed on the hot plate of a magnetic stirrer. On Stirring fish ensures sufficient mixing. The pistons come with Cotton plug closed. The flasks are under the boiling of the Nitric acid boiled for approx. 45 min. After cooling, they are approx. 5 min rinsed thoroughly with fully deionized water.

b) Instructions for hydrophobizing

The silanizing reagent dichloromethylsilane (SIGMA) is 5% (w / v) in Toluene dissolved. The hydrophilized flasks (see above) become about one Third of their nominal volume filled and about 5 minutes strong under the hood shaken so that the entire wall is wetted with a film of liquid becomes. The flasks are then emptied (not rinsed with water!) And for 24 hours air dried under the hood. Then the pistons are 5 min with fully deionized water rinsed and separated from other glass objects in the Drying cabinet dried.

Claims (13)

1. Culture medium for the cultivation of filamentous fungi, containing a) about 5 to about 20 [g / L] glucose, b) about 1 to about 6 [g / L] peptones as well c) about 0.006 to about 0.03 [g / L] CuSO ₄ . 2. Culture medium according to claim 1, containing a) about 5 to about 20 [g / L] glucose, b) about 1 to about 6 [g / L] peptones, c) about 0.006 to about 0.03 [g / L] CuSO ₄ , d) about 0.01 to about 1.0 [g / L] KH ₂ PO ₄ , e) about 0.0003 to about 0.003 [g / L] ZnSO ₄ , f) about 0.01 to about 0.8 [g / L] K ₂ HPO ₄ , g) about 0.00005 to about 0.005 [g / L] FeSO ₄ , h) about 0.009 to about 0.09 [g / L] MnSO ₄ and i) about 0.005 to about 0.5 [g / L] MgSO ₄ . 3. Culture medium according to claim 1 or 2, containing a) 7 to 18 [g / L] glucose, b) 2 to 5 [g / L] peptones, c) 0.009 to 0.022 [g / L] CuSO ₄ , d) 0.1 to 1.0 [g / L] KH ₂ PO ₄ , e) 0.0004 to 0.002 [g / L] ZnSO ₄ , f) 0.02 to 0.6 [g / L] K ₂ HPO ₄ , g) 0.0001 to 0.005 [g / L] FeSO ₄ , h) 0.02 to 0.07 [g / L] MnSO ₄ and i) 0.01 to 0.4 [g / L] MgSO ₄ . 4. Culture medium according to one of claims 1 to 3, containing a) 10 to 15 [g / L] glucose, b) 3 [g / L] peptones, c) 0.016 [g / L] CuSO ₄ , d) 0.6 [g / L] KH ₂ PO ₄ , e) 0.0006 [g / L] ZnSO ₄ , f) 0.04 to 0.4 [g / L] K ₂ HPO ₄ , g) 0.0003 to 0.003 [g / L] FeSO ₄ , h) 0.04 [g / L] MnSO ₄ and i) 0.02 to 0.2 [g / L] MgSO ₄ . 5. Culture medium according to one of the preceding claims, additionally comprising a compound which induces the biosynthesis of enzymes, in particular a compound which is selected from: a) ABTS® b) 3,4-xylidine, c) humic acids, d) caffeic acid, e) guaia cole, f) sinapic acid, g) o-toluidine, h) chelating lignin compounds, i) syringaldazine and preferably j) benzyl alcohol. 6. Process for the production of enzymes from filamentous fungi, characterized in that one a) cultivated filamentous fungi in a culture medium according to any preceding claim and b) the enzymes are isolated from the cultivated fungi or from the culture medium. 7. The method according to claim 6, characterized in that the filamentous mushrooms are selected from Genera Acremonium, Aspergillus, Coriolus, Coprinus, Pleurotus, Stachybotrys and Fusarium, especially among mushrooms of the genus Stachybotrys; preferably under Mushrooms of the species Stachybotrys atra, Stachybotrys aurantia, Stachybotrys bisbyi Stachybotrys chartarum, Stachybotrys parvispora, Stachybotrys kampalensis, Stachybotrys theobromae, Stachybotrys cylindrospora, Stachybotrys dichroa, Stachybotrys oenanthes and Stachybotrys nilagerica; particularly preferred among Stachybotrys bisbyi and Stachybotrys chartarum. 8. The method according to claim 6 or 7, characterized in that the enzymes are selected from a) proteases, b) oxidoreductases, such as oxidases, in particular phenol oxidases, peroxidases, laccases, tyrosinases; c) lipases and d) glycosidases such as amylases, cellulases and glucanases. 9. The method according to any one of claims 6 to 8, characterized in that you can cultivate filamentous mushrooms in one hydrophobized culture vessel, preferably in a hydrophobized glass Vessel, especially a hydrophobized glass bulb. 10. The method according to any one of claims 6 to 9, characterized in that a) one uses as filamentous fungi those of the species Stachybotrys bisbyi, and b) the enzyme to be obtained is a phenol oxidase, and c) one cultivates the mushrooms at a temperature of 26 ° C. 11. Use of a culture medium according to one of claims 1 to 5 for Cultivation of filamentous mushrooms. 12. Use of the immediate process products from a process according to one of claims 6 to 10 in detergents, cleaning agents, Hand washing detergents, hand dishwashing detergents, Dishwasher detergents, cosmetic or pharmaceutical preparations, hair tinting and hair dyes and agents for dyeing or bleaching Textiles, pulp, paper, furs, skins or leather. 13. Detergents, cleaning agents, hand washing agents, Hand dishwashing liquid, machine dishwashing liquid, cosmetic or pharmaceutical Preparations, hair tinting and coloring agents and dyeing preparations or bleaching textiles, pulp, paper, furs, skins or leather, containing immediate process products from a process according to one of claims 6 to 10.