According to the present method a cellulose-containing substrate is brought into contact with a reagent producing an oxoammonium ion in the presence of an oxidation agent. The reaction is preferably carried out in a liquid medium and the reaction product is separated from the medium after the reaction.

Pulp fibres contain cellulose as the major carbohydrate. By converting the primary alcohol groups of cellulose into carboxylic or aldehyde groups, the properties of cellulose can be modified. Thus, to mention an example, new carboxylic groups can be introduced into cellulose or a cellulose-based material by oxidation. Such carboxylic groups will affect the properties of cellulosic pulps; by increasing the carboxylic group content, the swelling and as a result the fibre flexibility can be increased. On an average, kraft pulps contain 30 to 150 mmol carboxylic groups/kg (3).

Carboxylic groups can be also generated onto different cellulose textile fibres, e. g. cotton and linen. in order to obtain novel properties for the end-product. For instance. the increase in carboxylic group content in cotton or linen will increase the water absorbency and subsequently affect fabric properties. Furthermore, by increasing the carboxylic group content in recycled fibres or in mechanical fibres the technical properties can be up-graded.

Nitrogen oxides have been used in chemical oxidation of carbohydrates (4). The disadvantage of oxidation with nitrogen oxides is that the method is not selective to primary alcohol groups. Also the secondary OH-groups are oxidized. which will cause depolymerization.

Semmelhack et al. (1983) showed that primary alcohol groups can be selectively oxidised

by a stable organic nitroxyl radical TEMPO. The actual oxidant is the oxoammonium ion, which can be obtained from TEMPO by chemical oxidation. Several oxidants e. g. hypohalites are able to oxidise TEMPO to oxoammonium ion. The oxidation of various natural occuring polysaccharides is discussed by Chang and Robyt (2).

To complete the survey it should be mentioned that Krishna et al (8) have studied studied the mechanism of superoxide dismutation by various 5-and 6-membered nitroxides. The superoxide was enzymatically generated.

The present invention is based on the idea that TEMPO can be enzymatically oxidized with, e. g. phenoloxidases such as laccases. to the oxoammonium ion. Therefore, laccases can be used to replace chemical oxidants which may be hazardous or even toxic. The present invention thus provides for oxidation of carbohydrates in cellulosic fibres by enzymatically oxidizing TEMPO.

More specifically, the present invention is mainly characterized by what is stated in the characterizing part of claim 1.

Considerable avantages are obtained by the present invention. A method for selective oxidation is provided. which ulves rise to the formation of carboxylic and carbonyl groups at desired ratios in the cellulose. By using a phenoloxidase as the regenerative oxidant it is possible to avoid the environmentallv harmfull halide-containing materials commonly used as oxidants. The final electron acceptor is oxygen or hydrogen peroxide, which clearly diminishes the formation of by-products in the reaction system and which will make it easier to separate and purify the product. Furthermore. when oxidative enzymes, such as a phenoloxidase. are used as regenerative oxidants the oxidation can be carried out at mild reaction conditions: room temperature and about neutral pH.

Next the invention will be examine more closely with the aid of a detailed description and with reference to a number of working examples. The attached drawing depicts schematicallv the reactions of TEMPO in a oxidation system. Figure I A shows the oxidation of TEMPO bv chemical oxidants and Figure 1B gives the corresponding

presentation of the situation according to the present invention wherein the oxidation is achieved bv enzymes.

As Figures 1A and 1B will show. the stable nitroxvlradical (species 1) is oxidized to the oxoammonium ion (species 2). which oxidizes the alcohol group (RCHOH) e. g. to aldehyde (RCHO), while the oxoammonium ion at the same time is reduced to hydroxylamine (species 3).

The regenerative oxidant used in the case of Figure lA is hypochlorite and in Figure 1 B oxygen. In the first case. a catalytic amount of bromide is introduced into the reaction. The bromide will be oxidized to hvpobromite which is a stronger oxidant than hypochlorite.

As mentioned above. different kinds of oxidative enzymes can be used. In particular. oxidative enzymes known to be capable of catalyzing oxidation of phenolic groups are used. These enzymes are often called phenoloxidases (E. C. 1.10.3.2 benzenediol : oxygen oxidoreductase) and they catalyze the oxidation of o-and p-substuted phenolic hydroxyl and amino/amine groups in monomeric and polymeric aromatic compounds. The phenoloxidases include oxidases and peroxidases."Oxidases"are enzymes which catalyze oxidative reaction using molecular oxygen as their substrate. whereas "peroxidases"enzymes which catalyze oxidative reactions using hydrogen peroxide as their substrate.

As specific examples of oxidases the following can be mentioned: laccases (EC 1.10.3.2), catechol oxidases (EC 1.10.3.1), tyrosinases and bilirubin oxidases (EC 1.3.3.5). Preferably, the enzymes used in the present invention are laccases. The laccases can be produced by white rot fungi. in particular by strains Trametes (formerlv Coriol2ls) versicolor or hirsuta or villosa. Other known producers of laccase are the strains of the following genera: Agariclls. Arn2illaria. Aspergillus. l3otrvtis. Fusariurm. Lentinus, Phlebia,Polyporus,Podospora,PycnoporusandMonocillium,Neurosp ora, Schizophyllum.

The invention is not. however. limited to the indicated origins of the enzyme nor to the

isolation method, and the enzyme can also be obtained by other methods.

Thus. it is possible to produce the fenoloxidase enzyme by microorganisms, which have been mutated or genetically constructed to produce the desired enzyme, or by other production host strains, to which the gene encoding this enzyme has been transfered.

The amount of laccase used can be about 1 to 1000 nkat/g cellulose. The oxidation can be carried out in liquid phase, because TEMPO is water-soluble. The temperature is about 10 to 70 °C, preferably about 20 to 40 °C and the pH is 3 to 9. The amount of TEMPO is 0.01 to 50 weight-%, preferably about 0.1 to 20 weigth-%, calculated from the amount of cellulose.

In practice, the reaction is carried out by dissolving a selected amount of TEMPO (in the form of the stable nitroxyl radical) into water; and suspending cellulose-containing substrate into the water phase to obtain a dry matter content of about 0.1 to 20 weigth-%. A buffering agent can be added to the water to adjust the pH of the reaction medium to a value suitable for the laccase treatment (preferably 4 or higher). After the addition of laccase oxygen is introduced into the reaction mixture either as (pure) gas or as an airstream while agitating the mixture. The duration of the oxygen introduction will take about 10 min to 24 hours. depending on the amount of substrate to be oxidized. After the reaction, the oxidized cellulosic material is separated from the liquid by filtration, it is washed and dried. if required.

As mentioned above. peroxidases can also being used as oxidative enzymes for oxidizing TEMPO. In that case. the final electron acceptor is hydrogen peroxide.

As a result of the oxidation according to the present invention. an oxidized cellulose- containing material is obtained in which a part of the primary OH groups have been converted to carbonyl and carboxylic groups.

The method can be used for oxidizing fibres, in particular fibres of natural sources, such as cellulose from annual or perennial plants or trees. These fibres typically contain some

residues of the other components present in the source of origin, such as lignin and hemicellulose, in the case of cellulose fibres from tree pulp, and pectins in the case of cotton. As specific examples of oxidation substrates, fibres obtained from mechanical. chemical, chemimechanical or recycled pulps can be mentioned. These kinds of oxidized pulps can be used for preparing paper or boards having improved properties, in particular improved paper technical properties, flexibility, WRV and strength properties.

The method can also be employed for oxidizing cellulosic textile fibres, yarns and fabrics. These materials typically comprise cellulosic textile fibres selected from cotton, linen, hemp, ramie and viscose. Oxidation of the materials will increase the caboxylic group content of the fibres, yarns and fabrics and thus affect the properties thereof.

Further TEMPO-based oxidation will provide fibres, yarns and fabric with modified properties selected from the group of handle, drapability, softness and water absorbency.

Finally, it should be noted that the use of traditional oxidants. such as hypochlorite and- bromide for oxidization of cellulose fibres (defined above) via TEMPO is not disclosed in the prior art.

The following non-limiting examples illustrate the invention.

Example 1 (reference) Chemical oxidation/hypochlorite and bromate and TEMPO mediated oxidation of ECF pine kraft pulp As a reference, ECF pine kraft pulp was chemically oxidized by TEMPO together with hypochlorite and hypobromite (generated from sodium bromide) as described by (2).

5 g of ECF pine kraft pulp was suspended in 180 ml of water. 50 mg of TEMPO, 1.6 g NaBr and 60 ml of 8 % (w/v) hypochlorite solution (pH was adjusted to 10.8 by NaOH) were added to the suspension. The reaction mixture was stirred at room temperature for 5 hrs. pH was checked and adjusted to 10.6 by NaOH. After reaction the ECF pine kraft pulp was collected by filtration. It was washed by 5 times volume (Sx250 ml) deionized

water and by 1 time volume 100 mM acatate buffer pH 5.0.

The carboxylicic group content of the TEMPO-oxidized pulp and the reference pulp was analyzed by conductometric titration. The amount of carbonyl groups present in the pulp was estimated from the FTIR spectra at wavelength 1720-1620 cm'.

Table 1. Carboxylic and carbonyl group content of the reference ECF pine kraft pulp and the oxidized ECF pine kraft pulp Pulp COOH groups, mmol/kg Carbonyl groups, relative units from FTIR Ref 30 1 TEMPO oxidized pulp 900 5 According to the results, chemical oxidation by TEMPO and hypochlorite/-bromate generated new carboxylic groups in ECF pine kraft pulp. Also the amount of carbonyl groups was increased by oxidation. The amount of carboxylic and carbonyl groups in the pulp were increased 30 and 5 times, respectively, by the TEMPO oxidation.

Example 2 Enzymatical TEMPO-mediated oxidation/Laccase + TEMPO mediated oxidation of ECF-kraft pulp Laccase + TEMPO mediated oxidation of ECF bleached pine kraft pulp was carried out at pH 5 with Trametes laccase (dosage 1000 nkat/g pulp). The reaction conditions were, consistency 1 %, pH 5* temperature room temperature. As a reference, the pulp was treated with laccase alone. TEMPO alone and without laccase or TEMPO. After the treatment the pulps were washed with distilled water. Carboxylic groups were measured by conducto-metric titration, carbonyl groups estimated by infrared spectroscopy and the carbohydrate composition was analyzed by HPLC after total enzymatic hydrolysis as described by (6). According to the HPLC analysis, the carbohydrates in the pulp were modified as compared to the reference. Also according to the FTIR analysis, the

modification was observed in the amount of carbonyl groups in the pulp.

TEMPO/laccase oxidation increased carbonyl groups in ECF pine kraft pulp about 25 %.

Example 3 Technical properties of the oxidized pulps The technical properties, especially the water retention value WRV, of the laccase- TEMPO-oxidized kraft pulp were measured before and after PFI-refining. The viscosity of the pulp was also measured according to SCAN-method. According to the results the properties were modified as a result of the increased amount of carboxylic and carbonyl groups in the pulp.

Example 4 Cotton fibres were oxidized with laccase and TEMPO and the effect of the treatment on the properties of the fibres were analyzed. Compared to the untreated fibres, the properties of the laccase-TEMPO-treated cotton were modified.

REFERENCES 1. de Nooy, A. E. J., Besemer, A. C. and van Bekkum, H. (1996) Highly selective nitroxyl radical-mediated oxidation of primary alcohol groups in water-soluble glucans.

Carbohydrate Research 269: 89-98.

2. Chang, P. S. and Robyt, J. F. (1996) Oxidation of primary alcohol groups of naturally occuring polysaccharides with 2, 2,6,6-tetramethyl-1-piperidine oxoammonium ion. J. Carbohydrate Chemistry 15: 819-830.

3. Lindstrom, T. and Carlsson. G. (1982) The effect of chemical environment on fibre swelling. Svensk Papperstidning 85: R14-20.

4. Maurer, K. ja Drefahl, G. (1942) J. Am. Chem. Soc. 64: 121.

5. Semmelhack, M. F., Chou, D. A. and Cortes, J. (1983) J. Am. Chem. Soc. 105: 4492.

6. Tenkanen. M., Hausalo, T., Siika-aho. M., Buchert, J. and Viikari, L. (1995) Proc.

8th Int. Symp. Wood and Pulping Chemistry, Helsinki 6-9 June 1995, Vol III: 189.

7. Heinzkill, M. (1995) Isolierung und Charakterisierung von Laccase und Peroxidasen aus Basidiomyceten der Ordnung Avaricales. Ph. D thesis University of Kaiserslautern Germany, p. 178.

8. Krishna et al., 1992, Proc. Natl. Acad. Sci. USA, Vol 89. pp. 5537-5541