BACKGROUND ART The most used tanning agents are chrome based, but chrome based tanning agents are considered environmentally unfriendly, and thus, alternative tanning agents, which are environmentally friendly, are always welcome.

In the tanning art it is intended to perform a fixation of tanning agents within the collagen matrix, both due to the fact that the quality of the leather will be better with increasing degree of fixation, and also because the pollution will be reduced with increasing degree of fixation and uptake of tanning agents. However, the prior art methods for tanning of hides by means of tanning agents are open to improvement in regard to degree of fixation.

US 3,212,996 describes a process for enzymatic modification of organic tanning agents. The tanning agents are vegetable or synthetic phenolic agents. The enzyme can be oxidase or peroxidase, and an oxidizing agent, e. g. hydrogen peroxide can be added in order to carry out the oxidase reaction.

WO 93/12259 describes a method for tanning of hides by means of tanning agents in a tanning bath, the bath comprises besides the hides an oxidoreductase, and as tanning agent an aromatic compound and possibly other compounds which are able to react with the hides and/or the aromatic compound, and that an oxidation agent is introduced into the equilibrated tanning bath. The modified tanning agents in US 3,212,996 are produced separately from the leather, whereas the modified tanning agents in WO 93/12259 are produced in situ on the leather.

SUMMARY OF THE INVENTION The object of the invention is the provision of a method for tanning of hides or skins by means of tanning agents, in relation to which the degree of fixation and uptake of tanning agents is similar or increased in comparison to the prior art methods of this kind.

It has been found according to the invention that it is possible to obtain at least the same degree of fixation in comparison to the prior art methods of this kind, if the tanning is supported by a laccase derived from the genus Myceliophthora in the tanning bath, and if specific primary and secondary substrates for this enzyme are added sequentially.

Thus, in its first aspect, the present invention provides a method for tanning of hides by means of tanning agents in a tanning bath is characterized by the fact that the bath besides the hides comprises a laccase derived from the genus Myceliophthora, and as tanning agent an aromatic compound and possibly other compounds which are able to react with the hides and/or the aromatic compound, and that an oxidation agent is introduced into the equilibrated tanning bath.

In a second aspect the invention relates to a tanning bath comprising a tanning agent and a laccase derived from the genus Myceliophthora.

In a third aspect the invention relates to the use of a tanning bath for tanning skins and hides.

A further advantage related to the invention is the fact that the use of toxic co- agents (like formaldehyde) used to crosslink organic tanning agents is avoided. Furthermore the invention is environmentally friendly, as cheap synthetic tanning agents can be used instead of the conventionally used tanning agents produced by extraction of wood from tropic forests.

DETAILED DISCLOSURE OF THE INVENTION The present invention provides a method for tanning of hides by means of tanning agents in a tanning bath, wherein the bath besides the hides comprises a laccase derived from the genus Myceliophthora, and as tanning agent an aromatic compound and possibly other compounds which are able to react with the hides and/or the aromatic compound, and that an oxidation agent is introduced into the equilibrated tanning bath.

Laccases Laccases (benzenediol : oxygen oxidoreductases) (E. C. class 1.10.3.2 according to Enzyme Nomenclature (1992) Academic Press, Inc) are multi-copper containing enzymes that catalyses the oxidation of phenols. Laccase-mediated oxidations result in the production of aryloxy-radical intermediates from suitable phenolic substrates; the ultimate coupling of the intermediates so produced provides a combination of dimeric, oligomeric, and polymeric reaction products.

The inventors have found that suitable laccases may be derived from a strain of Myceliophthora sp., e. g. M. thermophila described in WO 95/33836.

Tanning agents The primary substrate for the laccase is the aromatic compound, preferably a phenol, which is a vegetable or synthetic tan or tanning agent. Examples of vegetable tans which usually are polyphenol compounds extracted from plant materials are catechol tans (or so-called condensed tans) and pyrogallol tans (or hydrolysable tans) as described in Part 3, Tanning Processes, in Leather Technician's Handbook, J. H. Sharphouse (Ed), Leather Producers Assoc., Kings Park Road, Moulton Park Northhampton, NN31 JD, 1989. Examples of synthetic tans include, but are not limited to, mono-or oligomeric aromatic or aliphatic compounds, optionally substituted with one or more of the following functional groups:- halogen,-OH,-SO3H-,-COOH,-NH2,-OCH3, or aryl, alkyl, alkene each of which may also be substituted as above. Further compounds, which may be used as tanning agents include heterocyclic and aliphatic compounds with the same functional groups as above indicated.

Specific examples of aromatic compounds which may serve as primary substrates for the laccase in the present invention include : catechol, aniline, tyrosine, catechin, aromatic amino acids and derivatives thereof, pyrogallol, gallic acid, ferulic acid, 2-methyl-1 napthol, 2,6- disubstituted monophenols, guaiacol, coniferyl alcohol, protocatechuic acid, resorcinol, hydroquinone, caffeic acid, m-cresol, p-coumaric acid, anisidine, syringaldazine and syringaldehyde.

The oxidizing agent which is the secondary substrate for the laccase is oxygen.

The hides and the tanning bath may be kept under conditions with as low an oxygen concentration as possible in order to prevent uncontrolled polymerization prior to a sufficient penetration of the laccase and the tanning agent into the hide matrix. In the equilibrated tanning bath the laccase and tanning agent has penetrated into the hide matrix to such degree that an equilibrium condition is obtained.

The method according to the invention is in particular performed by first applying the tanning agent (s) and thereafter the laccase to the hide as separate solutions. In another embodiment the tanning agent (s) and the laccase is applied to the hide as a mixture.

Depending on the types of substrate and on the desired characteristics of the hide, the polymerization process is carried out in a pure aqueous system or in solvent systems, the solvent (s) being water miscible or water immiscible. When using a water containing system the pH may be controlled by employing conventional buffers (e. g. phosphate, carbonate/bicarbonate, tartrate, carbonate, or acetate buffers). The polymerization process may be further controlled by metering the addition of oxygen. The enzymatic reaction may be terminated by chemical or thermal inactivation of the enzyme.

The enzymatic reaction may be carried out at temperatures between 5 and 95°C.

In a particular embodiment the reaction is carried out at the optimum temperature of the enzyme, at a pH in the range from 3.0 to 9.0, particular in the range from 4.0 to 8.0, more particular in the range from 6.0 to 8.0.

Process conditions may be additionally optimized by varying the relative concentrations of the organic and inorganic substrates, polarity and concentration of solvent, enzyme concentration etc. Preferably, the enzyme is added in an amount of 0.001 to 0.1%, more preferably 0.005 to 0.05% based on the weight of pelt to be treated.

The present invention makes possible a process for tanning by allowing the use of new and improved tanning agents. Thus, new and/or additional functionalities (e. g. positively or negatively charged substituents) may be added to hitherto known or unknown tanning agents in order to improve hide characteristics such as: water retention, shrinking temperature or degree of fixation within the collagen matrix.

A particular embodiment of the method according to the invention is characterized by the fact that the tanning agent is a synthetic or vegetable, aromatic tanning agent. In this manner a high quality leather can be produced, especially a leather with high shrinking temperature.

A particular embodiment of the method according to the invention is characterized by the fact that the tanning bath is aqueous. In this manner the tanning can be performed in an environmentally friendly manner.

A particular embodiment of the method according to the invention is characterized by the fact that the tanning bath is organic. In this manner an improved polymerization and thus an improved degree of fixation can be obtained.

Mediators In the present context, the term"mediator"is intended to mean an agent capable of acting as a substrate of laccase, and includes compounds commonly referred to in the art as"modifiers", as well as"enhancing agents".

Examples of mediators capable of enhancing the activity of oxidoreductases include the compounds described in WO 95/01426, which is hereby incorporated by reference, and represented by the general formula I : Specifically contemplated compounds within the above formula I include the following : 2,2'- azino-bis (3-ethylbenzothiazoline-6-sulfonate (ABTS); 6-hydroxy-2-naphtoic acid; 7-methoxy-2- naphtol ; 7-amino-2-naphthalene sulfonic acid; 5-amino-2-naphthalene sulfonic acid; 1,5- diaminonaphthalene ; 7-hydroxy-1,2-naphthimidazole; 10-methylphenothiazine ; 10-phenothia- zine-propionic acid (PPT); N-hydroxysuccinimide-10-phenothiazine-propionate; benzidine; 3,3'- dimethylbenzidine ; 3,3'-dimethoxybenzidine; 3,3', 5,5'-tetramethylbenzidine; 4'-hydroxy-4- biphenylcarboxylic acid; 4-amino-4'-methoxystilbene ; 4,4'-diaminostilbene-2, 2'-disulfonic acid; 4,4'-diaminodiphenylamine; 2,7-diaminofluorene; 4,4'-dihydroxy-biphenylene; triphenylamine ; 10-ethyl-4-phenothiazinecarboxylic acid ; 10-ethylphenothiazine ; 10-propylphenothiazine ; 10- isopropylphenothiazine ; methyl-10-phenothiazinepropionate ; 10-phenylphenothiazine ; 10-allyl- phenothiazine; 10-phenoxazinepropionic acid (POP); 10- (3- (4-methyl-1-piperazinyl) propyl) phe- nothiazine; 10- (2-pyrrolidinoethyl) phenothiazine; 10-methylphenoxazine ; iminostilbene ; 2- (p- aminophenyl)-6-methylbenzothiazole-7-sulfonic acid; N-benzylidene-4-biphenylamine ; 5- amino-2-naphthalenesulfonic acid; 7-methoxy-2-naphtol ; 4,4'-dihydroxybenzophenone; N- (4- (dimethylamino) benzylidene)-p-anisidine ; 3-methyl-2-benzothiazolinone (4- (dimethyl- amino) benzylidene) hydrazone ; 2-acethyl-10-methylphenothiazine ; 10- (2- hydroxyethyl) phenothiazine; 10-(2-hydroxyethyl) phenoxazine; 10- (3- hydroxypropyl) phenothiazine; 4,4'-dimethoxy-N-methyl-diphenylamine, and vanillin azine.

Other mediators contemplated include 4-hydroxybenzoic acid, L-tyrosine, syringate acids, ferulic acid, sinapic acid, chlorogenic acid, caffeic acid and esters thereof.

Still further examples include organic compounds described in WO 96/10079, which is hereby incorporated by reference, and represented by the general formula)) :

Specific compounds covered by the above formula 11 are acetosyringone, syringaldehyde, methylsyringate, syringic acid, ethylsyringate, propylsyringate, butylsyringate, hexylsyringate, octylsyringate and ethyl 3- (4-hydroxy-3, 5- dimethoxyphenyl) acrylate.

The invention will be illustrated by the following examples showing tanning with Myceliophthora thermophila laccase.

EXAMPLES Example 1: Tanning of cattle hides with use of vegetable tannins In the following example the weight calculation are based on % on split weight. The cattle hides are treated according to the following steps.

Wash: 200 % Water 30° C 15 min Drain until empty Deliming : 50 % Water 30-35° C 1-1.5 % Decaltal ES liq. * 30 min + 0.2 % Bascal S* 30 min PH 7-9 + 40 % Water 30-35° C 0.4-0.6% Novocor B1300*** 45 min

Drain until empty Wash: 200 % Water 15-20° C 10 min Drain until empty Pickl.-Tanning : 60-70 % Water 20°C 6-7 % Salt 15 min + 0.8 % Formic acid 10 min + 0.7 % Sulfuric acid 60-90 min PH 2.8-3.2 + 10 % Mimosa 5 % Quebracho 0.2 % Cartan O** 120min + 0.1-0.05% Myceliopthora thermophilia Laccase (250 LAMU/g) 10-15 times lower dosage of Methylsyringate compared to the enzyme dosage 540 min PH 4. 0-5.0 * Tradename of BASF ** Tradename of Cariant *** Tradename of Novozymes Shrinkage temperature with the laccase is similar to glutardialdehyde treated leathers.

Example 2: Retanning with Myceliophtora laccase The hides from example 1 are treated according to the steps below. In this example the weight calculation are based on % on shaved weight.

Neutralization : 150 % Water 40°C 2.5 % Sod. formate 2.0 % Sod. bicarbonate 90 min. PH 5.5 Drain until empty Wash: 200 % Water 40°C 10 min.

Drain until empty Retanning: 100 % Water 40°C 3.0 % Relugan RV* 15 min.

+ 1.5 % Sod. Bicarbonate 30 min. PH 5. 0 + 0.1-0.05% Myceliopthora Thermophilia Laccase (250 LAMU/g) 10-15 times lower dosage of Methylsyringate compared to the enzyme dosage 120 min + 3.0 % Mimosa 4.0 % Chestnut 1.0 % Retingan R7** 120 min.

+ 0.5 % NH40H 10 min.

+ 3.0 % Dyestuff 60 min + 8.0 % Densodrin CD* 80 min.

+ 1. 0 % Formic acid 20 min.

+ 1.0 % Top dye 20 min.

+ 1.5 % Formic acid 20 min.

+

200 % Water 60°C 4.0 % Chromosal B* 90 min.

Drain until empty Wash: 200 % Water 60° 10 min.

Drain until empty 200 % Water 60°C 3.0 % Densodrin CD* 20 min.

+ 1.0 % Chromosal B* 40 min.

+ 0.5 % Formic acid 10 min.

*Tradename of BASF ** Tradename of BAYER In crust condition the leather treated with laccase show a more compact aspect what indicate that more vegetable tannins have been bounded to the leather fibers. The grain is fine and tight.

Determination of Laccase Activity (LAMU) The LAMU method is used for determining the activity of Myceliophthora thermophila lac- case. 1 laccase unit (LAMU) is the amount of enzyme which catalyses the conversion of 1.0 micro mole syringaldazine per minute under the analytical conditions to be found in WO 98/40471, pages 18 to 20.