AU663501B2 - Process, using enhanced-action laccase enzymes, for the delignification or bleaching of lignocellulose-containing material or for the treatment of waste water - Google Patents

Description

OPT DATE 30/12/92 A0JP DATE 11/02/93 APPLN. ID 17933/921111 1111111 PCT NUMBER PCT/EP92/01086 III iIiI i m AU921 793 I IN IL IN NA I IUN NA LL ANINVI I;LI)U INkU VtKU1 LIN I I-I L, Ni I 11 IJ \1 VI I KAO/U U51. IJ II~ I-1 INTERNATIONALE ZLISAMNIENARBF.IT AUF D)EM CiEI3117ET l)ES PATE NTI:,S ENS (PCT) (51) Internationale Ilaentkassifikation 5 D2 IC 5100, 9/10, 5/02 C02F 9/00 AlI (11I) Internationale V 7 eriiffenflichuitgsnumimer: (43) Interriationales Ver6ffentlichungsdaturn: 26. No\ WO92/20857 embl er 1992 (26.11.92) (21) Internationales Aktenzcicl (22) Internationales Annmelded IPrioritiitsdaten: P 41 16 133.5 1P4 1 37 761.3 13CT EP392 01086 IS. M~ai 1992 (18.05,92) 17. Mai 1991 (17.05.91) 1DLF 16. Noxember 1991 (16.11.91) D)E (81) Best i mrn u ngsstaa ten :AT (eu roliisclle Patent). AL 13L (europalisches Patent), 13K (europfikiche. Patent). I)E (europ~iischic. Patent). I)K (eU ropaischc Patent). ES (eurnpiiisches Patent). Fl. F-R (europaikche' Patent), G 13 (europ~isches Patent). G R (europ'lii,,he, Patent). iilu. IT (europ~iiches Patent). J P. LU I europ~iisches Patent). MIC (europjiisches, Patent). N L (europliisches Patent). NO. R U, SEL (eu rop~iischcs Patent). L.

Verliffentlichit AMU ii!Lrflatjnlem Recherc/tenbericli r I lAlaif tier flr 4nderien/ der .lospriklic :11VC/ec/titLei ErA i. Iv'rlffioliclintg ivird iLcL'rhlt h f f fil dcrim (71)(72) Annielder tind Erfinder: CALL. Hans-Peter [DE 'DE]: Heinsbergoer Str. 14a. D-5 132 Ubach-Palenbero (DE).

(74) Arsalt: FITZNE:-R FITZNER: Amn Eichfr~StChen 2a. D)- 4030 Ratinaen-Lintorf (DE).

&~635O1 (54)Title: PROCESS. USING ENHANCEI)-ACTION LACCASE ENZYMES. FOR THE I)ELIGNIFICATION OR B3LEA- CHING OF LIGNOCELLULOSE-CONTAINING MATERIAL OR FOR THE TREATMENT OF W\ASTE WA-

TER

(54) Bezeichnung: VERFAHREN ZUR l)FLIGNIFIZIERUNG VON LIGNOCELLULOSEHALTIGEM M1ATERIA\L.

BLEICHE UND I3EHANDLUNG VON AI3WASSERN NIITTELS LACCASEN MIIT ERWEITERTI-R VI RKSAM KEIT (57) Abstract The invention concerns a process for the removal of lignin from lignocellulose-containing material, the bleachitng of lignocellulose-containing material the enzymatic dc-inking of recycled paper) and/'or the treatmetnt of wvaste wvater arising fromn the manufacture of paper or celltulose. The process Uses laccase enzymes obtained fromn CorioILus versicolor or other laccase producers, at the same time adding, or metering in non-aromnatic redox agents plus phenolic and 'or non-phenolic aromatic redox comnpounds. the phenolic and non-phenolic units of the lignin either being oxidized directly by the actiotn of these phenolic and or non-phenolic aromatic compotinds. or tie lignin being oxidized b\ other phenolic and 'or tion-phenolic compounds produced b\ the oxidizing actiotn of these compounds.

(57) Zusarnnenfassung Die vorliegende Erfindune betrifft emn Verfahren 7timi Abbau \von Lig-nin auIs lignioceiL1,llulshltigctnfl Nl aterial und oder lBleiche von lignlocellulosehaltigem Mlaterial ztmtn enzvmatischen Deinken von Altpapier und /.ur Behandlung \on PapierfabrikatiotiS- tind Zellstoiffabrikationsabwiisserni. wobei auIs Coriolus versicolor tind anderen Laccasebi Idnern ges onnene Laccasen onlter gleichizeitiger Zugabe Und 'oder Zuidosage von nicht-aromnatischcn Reduktions- und Oxidationsmitteln sowie solchen phetiolischen Verhindungen Lind 'oder nicht phenolischen Aromaten eiingesetzt xs erden. die entweder die lihenolisehen und nicht phenolisehen StrUktUrcn des lignins direkt durch die \Virkung dieser phenolischen und oder nicht-pheriolischen Aromnaten omidieren ocler m ittels dureb diese \'erbindungen vermittelter Oxidatioti weiterer phenolischer uind 'oder nichit phetiolischer Verbinduingen dais Lietint oxidiereti.

A process for the delignification of material containing lignocellulose, bleaching and treatment of white water by means of laccases with extended efficiency The present invention relates to a new process for the delignification of material containing lignocellulose and/or bleaching of material containing lignocellulose for enzymatically deinking waste paper and for treating white water, in particular pulp and paper mill waste.

I. Prior art of chemical processes for the pulp manufacture, use of lignolytic enzymes: biopulping, other applications A. Chemical processes for the pulp manufacture and manufacture of wood pulps The sulphate and the sulphite processes are mainly used for the pulp manufacture today. With both processes pulp is produced by cooking and under pressure. The sulphate process employs the active chemicals NaOH and Na2S, the sulphite process uses Ca(HSO3)2 S02.

Moreover there are a few lower-polluting cooking processes with the help of organic solvents.

All processes have one principal aim: the lignin removal from the used plant material, wood or annual plants. The lignin which is the main component of the plant material (stalk or stem) must be removed since otherwise it is not possible to manufacture the non-discoloring paper which can be highly loaded.

1 i 2 The wood pulp manufacture processes employ stone grinding devices (groundwood) or refiners (TMP) which fibrillate the wood after corresponding pretreatment (chemically, thermally or chemo-thermally) through corresponding grinding or refining.

These wood pulps still possess a major part of the lignin and are above all used for the newspaper print and for the print of magazines or advertisements, here as coated paper in general.

B. Biopulping The mechanism of action of lignolytic systems has only been investigated for a few years when it was achieved that sufficient enzyme amounts could be obtained from the white rot fungus Phanerochaete chrysosporium through appropriate cultivation conditions and inductor additions. Enzyme species were found in this fungus: lignin peroxidases and manganese peroxidases which had been unknown up to then. Since this fungus in-vivo is a highly effective lignin degrader it was at first concluded that the lignin would also be degraded if the enyzmes were used without the fungus. This idea proved to be wrong since the enzymes, among others, lead to a repolymerisation of the lignin and not to the degradation thereof.

The same also applies analogously to other lignolytic peroxidases and to other lignolytic enzyme species such as laccases which degrade the lignin in an oxidative manner with the help of 02 instead of H202. In all cases it comes to similar processes during the degradation, that is to a radical formation which, if these radicals are not trapped in any form, react again with each other and thus repolymerise.

r t 37 3 So one was forced to return to in-vivo systems with respect to biopulping.

The fungus was brought into contact with wood chips, for example, so as to achieve a partial delignification which at least increased the bleachability and reduced the energy necessary for the mechanical aftertreatment (fibrillation).

Biopulping is generally understood to be the manufacture of pulp or pulp-like materials performed by means of biologically enzymatic processes or preliminary stages. With biopulping, it is above all tried to cultivate strains which do not form any cellulases, so-called cellulase-less-mutants, since only the lignolytic enzymes are desired, the white rot fungi, however, also form cellulases in the presence of lignocellulose. The cellulases would lead to unwanted yield losses through cellulose degradation in the case of the pulp.

A further difficulty is that the white rot fungi cannot use the lignin as a carbon source and thus are dependent on the cellulose and hemicellulose respectively as a carbon source.

Presently these difficulties are eluded in that the substrate is impregnated with an additional carbon source, for example glucose among others, so as to enable the cellulase-less mutant a growth at all.

In this connection, the main difficulty is the very slow rate of degradation of the lignin (days to weeks) so that in the case of the in-vivo process: wood chop chippings cellulaseless mutants (Phanerochaete chrysosporium) one is busy with the screening of new better wild strains among others. The final goal is to achieve a quicker and more effective lignin degradation in order to improve the batch as already mentioned above: reduction of the refining or grinding energy and improved bleachability.

i i -4 If it were possible to keep the cellulose and hemicellulose losses low one would achieve a high-yield material which, however, in contrast to the wood pulps manufactured by means of refining or grinding so far could, among others, be manufactured essentially poorer in energy, would be easier to bleach and would show better mechanical characteristics.

As mentioned the principal drawbacks remain: 1. the very slow lignin degradation 2. the only very restricted lignin degradation (few 3. probably inevitable apportioning of additional carbon source 4. difficulties caused by the process control such as great batch volume due to the slow growth and degradation times, by special aeration, pH-maintenance and freedom of contaminations.

There are also batches with cell-free enzyme syscems with respect to Phanerochaete chrysosporium.

However, here the application consists 1) in the bleaching of "kraft pulp" (that is the partially delignified pulp which results subsequent to the sulphate cooking with about 5-8 residual lignin content), 2) in the improvement of the mechanical characteristics of mechanical pulps, i.e. pulps which result from the mechanical-thermal or chemo-mechanical or chemo-thermo-mechanical treatment of wood pulps, such as TMP (thermo-mechanical pulp, CMP (chemo-mechanical pulp) and CTMP (chemo-thermo-mechanical pulp) and 3) in the decolorization of El white water (such which are obtained after the first bleaching stage of kraft pulp).

Here the application is restricted to the degradation of 0 t W y\ 7«

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5 lignin-percentage fractions (in the case of kraft pulp) up to a few percent (groundwood) within 12 to 48 hours. The bleaching is unsatisfactory as well.

Recently some experiments have been carried out in order to achieve better bleaching results with the help of haem compounds, such as haemoglobin as well, which, in a way, reproduce the active haem component in the ligin peroxidases.

Here the compounds are generally too expensive for an industrial use and partly also have undesired accompanying effects such as degradation of the cellulose.

Also species of another enzyme group, the hemicellulases, here the xylanases among others, are used as bleaching enzymes since here an effect on the bondings between hemicelluloses (xylans) and the lignin is assumed.

Also here the enzyme takes long to act with a relatively low bleaching effect and a lacking lignin removal which is a considerable drawback.

Further applications of the lignolytic enzymes are the decolorization and detoxication of pulp waste and the oxidation and consequently detoxication of chlorinated aromatics and other compounds. In this field there are presently no satisfactory solutions of the problem either since in decolorization and detoxication experiments either free or immobilized fungi in in-vivo fungus systems have been employed so far. Only over longer periods (days) results were achieved with satisfactory degradation and oxidation values respectively.

a fre-rre4 It is nowAAe object of the present invention to make available processes for the degradation of lignin from -6material containing lignocellulose and/or bleaching of material containing lignocellulose and for enzymatically deinking waste paper and for treating pulp and paper mill waste which does not have any of the aforementioned drawbacks.

According to the present invention there is provided a process for the degradation of lignin from material containing lignocellulose and/or bleaching of material containing lignocellulose for enzymatically deinking waste paper and for treating pulp and paper mill waste, characterized in that laccases obtained from Coriolus versicolor and other laccase formers are used by simultaneously adding and/or apportioning non-aromatic reducing and oxidizing agents as well as such phenolic compounds and/or non-phenolic aromatics which either oxidize the phenolic and non-phenolic lignin structures directly through the effect of these phenolic and/or non-phenolic aromatics or which oxidize the lignin through oxidation brought about by these compounds of further phenolic and/or non-phenolic compounds.

It is true that German patent applications DE 3636208 (WC 88/03190), P 408893.6, P 408894.4 describe the use of lignolytic enzymes while simultaneously apportioning reducing and oxidizing agents and other red/ox materials (salts etc.).

However, the main difference in comparison to these documents is the extended use of laccases, i.e. laccases which are considerably extended in their activity by the addition of certain substances (phenolic aromatics, non-phenolic aromatics) and thus can attack the entire lignin molecule and act like a lignin peroxidase.

According to the invention laccase above all of Coriolus versicolor is used. This laccase is used together with laccase substrates (phenolic compounds and non-phenolic compounds respectively) and/or phenol compounds and/or nonphenolic aromatics whereby these compounds are oxidized, then directly oxidize lignin as red-/ox-mediators on their part or Y w ll950811,p:\oper\dab,17933.spe,6

I~

7 effect the oxidation by means of interposed further red-/oxmediators (phenolic compounds or non-phenolic aromatics, such as veratryl alcohol, for example). Thus it is effected that laccase which only attacks the phenolic components of the lignin (they comprise only about 1/3 of the substructures) can also attack the non-phenolic substructures and is thus theoretically capable to degrade the entire lignin molecule completely, consequently acts just like a lignin peroxidase.

Thereby, these red/ox systems must "match" each other such that like in the respiratory chain the oxidized substance acts as oxidizing agent for the next substance and is reduced by it. Fig. 1 shows the mechanism of action of the laccases on the level of the prosthetic group (Cu 2 Cul+).

Fig. 1 2(Cu 2 laccase phenol 2(Cu+) laccase radical 2H+ 2(Cu+) laccase 1/2 02 2H+ 2(Cu 2 laccase

-I

OH 0 1 I H, e The laccase catalyses the formation of a mesomeric aryloxyradical from phenol.

In contrast to the processes described in the above-mentioned r- 8 patents for the use of laccase, a main advantage is the essentially simplified cultivation of Coriolus versicolor in contrast to the harder cultivable lignin peroxidase formers, above all Phanerochaete chrysosporium, in bigger fermentation vessels. The own yields are with a special inductor at about million IU/1. Only with the extension of the laccase effect on the quasi-specificity of a lignin peroxidase a really commercial use of these enzymes is achieved which is a considerable advantage. Lignin degradation experiments with laccase alone without the above-described system with laccases and red-/ox-mediators of phenolic or non-phenolic kind arranged between lignin and enzyme) with spruce/wood pulp as substrate with an initial lignin content of about 27 show a delignification up to a maximum of 22 residual lignin content, i.e. about 19 degradation experiments with the new system presently show a lignin degradation of up to 16 residual lignin content, i.e. about 41 i.e. more than double.

By adding reducing and/or oxidizing agents, salts and/or phenolic compounds a redox potential of 200-600 mV is adjusted according to the invention. It can be determined by means of a red-ox-electrode and maintained constant by means of a control unit and controller during the entire reaction time by adding oxidizing and reducing agents, salts and phenolic compounds in the process according to the invention.

Preferably air, 02 or hydrogen peroxide are used as oxidizing agents. Sodium bisulphite, ascorbic acid, dithionite and thiol compounds come into consideration as reducing agents.

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IViiz; ^r) 9 Metal salts are preferably used as salts. Particularly FeS04, FeC13, MnS04, Ti02, CuS04 and Mn3-acetate are possible here.

Moreover it is referred to the fact that the addition of complex formers is preferred according to the invention. For example ethylenediamine tetraacetic acid (EDTA) or diethylenetriamine pentaacetic acid (DTPA) are used as such.

In addition to the substances mentioned further substances can be added to the aqueous solution containing pulp.

Possible as such are NaOC1, detergents and polysaccharides.

Among others non-ionic, ionic, anionic, cationic and ampholytic surface-active ag nts as well as gi'.cans and xanthans belong thereto. It is further possible to use hemicellulases and/or pectinases and/or lipases in addition to the lignolytic enzymes, too.

The main difference compared to the study "Oxidation of nonphenolic substrates" (FEBS 267, 1990, pages 99 to 102) of R.

Bourbonnais and M.G. Paice lies, among others, in the completely different process control by simultaneously apportioning reducing and oxidizing agents in order to prefer the depolymerisation of the lignin through "trapping radicals" and suppression of the repolymerisation while simultaneously maintaining the reaction by means of supplying oxidizing agents. It is known that laccases also act with H202 and that the oxidizing action of the laccase is intensified by peroxide with certain substrates Blaich, Analytische Elektrophoreseverfahren (Analytic Electrophoresis Processes), Thieme-Verlag 1978, p. 59).

The system according to the invention is either used with laccase alone or with lignin peroxidases and/or manganese peroxidases of Phanerochaete chrysosporium, for example, or together with other white rot fungi. In the case of the 10 latter, lignin peroxidase (manganese peroxidases) laccase, it is worked with a pH-value of about 4-5 where both enzyme species are still active. The actual lignin degradation, the bleaching (with lignin degradation) or the swelling process during the enzymatic dei:king is effected by means of simultaneously apportioning reducing and oxidizing agents, i.e. through trapping of radicals which arise from the lignin degradation. Often a simultaneous presence of certain metal salts is important, such as Fe 2 Fe3+, Cu 2 Ti 3 of NaCC1 etc. which lead to certain radicals as to the "OH-radical (Fe+ 2 and/or Ti+ 3 H202), as to the 0 02H-radical (Ti 3 and 02) or to the singlet oxygen (NaOCl H202 NaCl H20 C2 which, on their part, can accelerate the lignin degradation. Also the presence of polysaccharides which are partly produced by the fungus itself or come from yeast cell walls which result between white rot fungus and yeasts in mixed cultures, and the presence of certain detergents which allow a better penetration of the enzyme between the fibres when delignifying and/or bleaching and which are obligatory for the foam and collector effect of the ink particles, are important aids. In the case of deinking, an additional bleaching system such as formamidine sulphinic acid (FAS)/NaOH can be important as well in order to compensate the bleaching action of NaOH/H O existing in the chemical system, and moreover the addition of soaps alone or combined with detergents can be important as a collector.

When enzymatically treating wood pulp waste (TMP/groundwocd etc.) laccase phenol or non-phenolic aromatics are used.

Through the well-aimed use of certain phenols or non-phenolic aromatics without adding reducing agents but while continuously supplying oxidizing means (H202, 02 or atmospheric oxygen), a nearly complete polymerisation of lignins contained in the white water is possible here, which 11 exceed the achievable values by far (20-50 when using laccases on their own. Thus it is possible to remove 70-90 of the lignin from the white water by rendering insoluble and therefore flocculating, sedimenting, filtering, microfloating etc. and to lower the CSB drastically up to 60-70 A. Example 1) Example: Enzymatic bleaching of sulphate pulp g oven-dry pulp (sulphate pulp) are stirred in a stirrer vessel at 1 stock density at about 500 rpm and 40*C. The pH-value is adjusted to pH 5 with 1 n HC1. 0.1-1.5 H202 based on the oven-dry substance, about 0.1 ETDA or DTPA, 0.01 -0.5 copper(II)sulphate based on the oven-dry substance are added. After adding 500 8000 IU (1 IU conversion 1 pm syringaladazine min nl enzyme) laccase of Coriolus versicolor about 2 x 10 3 1 x 10 veratryl alcohol and 0.01 -0.1 naphtol per oven-dry substance are added and the bleaching process is started while simultaneously dosing H202 and sodium sulphite solution. In doing so the redox potential of about 400 mV is maintained.

After the process is initiated it is continued for 2-4 hours.

The control of the process is carried out via a redox electrode and a pump control.

B. Example 2) Delignification of wood pulp (TMP) 12 1 g oven-dry TMP (1 in 100 ml water is adjusted to pH with 1 m HC1. 8 pmol veratryl alcohol, 400 pmol H202 and 0.01 0.1 naphtol per oven-dry substance with 1 m HC1 are added. As apportioning (1 ml/h) sodium bisulphite and H202 are simultaneously added as solutions (9.80 mmolar each). The reaction is initiated by adding about 100 000 IU enzyme (laccase of Coriolus versicolor). The reaction time is 4 hours at 40*C under about 500 rpm stirring.

C. Example 3) Example: Enzymatic deinking with pretreatment 100 g oven-dry waste paper (50 paper 50 magazines) are torn into 1 x 3 cm pieces. Afterwards 2 1 water are added (about 45 C) and the pH is adjusted to 7 7,5 ',ith alum.

Then 80-8000 IU laccases (1 IU conversion of 1 pmol syringaladazine min ml enzyme 8 pmol veratryl alcohol and 0.05-0.6 detergents as collectors and 0.2-2 pl sodium hydrogen sulphite solution (38-40 based on g oven-dry waste paper material each are added. Then it is refined at 3000 rpm in the desintegrator for 15 minutes. The waste paper material is then allowed to stand at about 45*C for 75 min or immediately floated in a commercial 20 1 floatation cell for 10-20 min.

D. Example 4) Enzymatic treatment of wood pulp waste (TMP, groundwood and others) Polymerisation of the lignin present.

250 ml unfiltered TMP white water is added to 10-200000 IU laccase per 1 0.01 x 10-3 0.1 naphtol and incubated while stirring and supplying atmospheric oxygen at 20-50°C 13 for 1-4 hours. For the use of laccase alone, a considerably increased polymerisation of the lignin present occurs which becomes insoluble and can be removed by the addition of apt flocculants such as polyethylene imines etc. or by microfiltration, centrifugation or filtration.

Claims (12)

14- THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. A process for the degradation of lignin from material containing lignocellulose and/or bleaching of material containing lignocellulose for enzymatically deinking waste paper and for treating pulp and paper mill waste, characterized in that laccases obtained from Coriolus versicolor and other laccase formers are used by simultaneously adding and/or apportioning non-aromatic reducing and oxidizing agents as well as such phenolic compounds and/or non-phenolic aromatics which either oxidize the phenolic and non-phenolic lignin structures directly through the effect of these phenolic and/or non-phenolic aromatics or which oxidize the lignin through oxidation brought about by these compounds of further phenolic and/or non-phenolic compounds. 2. The process according to claim 1, characterized in that lignin peroxidases and/or manganese peroxidases are used in addition to the laccases. 3. The process according to claim 2, characterized in that lignin peroxidases and/or manganese peroxidases from Phanerochaete chrysosprium or other white rot fungi are used. 4. The process according to any one of claims 1 to 3, characterized in that hemicelluloses and/or pectinaes and/or lipases are further added to the reaction solution. 5. The process according to any one of claims 1 to 4, characterized in that the pH-value is adjusted between 3 and 8. 6. The process according to any one of claims 1 to characterized in that the temperature is adjusted between and 60 0 C. T9 9 95081 l,p:\oper\dab,17933.spe,14 .7t 7. The process according to any one of claims 1 to 6, characterized in that a redox potential is adjusted between 200 and 600 mV. 8. The process according to claim 7, characterized in that metal salts are further added to the reaction solution. 9. The process according to claim 8, characterized in that FeSO 4 FeC1 3 MnS0 4 Ti02, CuSO 4 and Mn 3 -acetate are used as metal salts. The process according to claim 9, characterized in that NaOC1 is used. 11. The process according to any one of claims 8 to characterized in that sodium bisulphite, sodium dithionite, ascorbic acid, thiol compounds or gluthatione are used as reducing agents. 12. The process according to any one of claims 8 to 11, characterized in that air, oxygen, H202 or organic peroxides are used as oxidizing agents. 13. The process according to any one of claims 1 to 12, characterized in that detergents are additionally used. 14. The process according to claim 13, characterized in that non-ionic, ionic, anionic, cationic and ampholytic surface- active agents are used as detergents. The process according to any one of claims 1 to 14, characterized in that polysaccharides are further added to the reaction solution.

16. The process according to claim 15, characterized in that glucans, mannans, dextran, laevan or plant gums and/or polysaccharides which are formed by the fungi themselves or 95081 ,p:\oper\dab,17933.spe,15 16- produced with yeast in the mixed culture are added as polysaccharides.

17. The process according to any one of claims 1 to 16, characterized in that complexing agents are further added to the reaction solution.

18. The process according to claim 17, characterized in that ethylenediamine tetraacetic acid (EDTA) or diethylenetriamine pentaacetic acid (DTPA) are used as complexing agents.

19. The process according to any one of claims 1 to 18, characterized in that water glass is added.

20. The process according to any one of claims 1 to 19, characterized in that soaps, saponified fatty acids for instance, are added.

21. The process according to any one of claims 1 to characterized in that formamidine sulphinic acid (FAS)/NaOH is added to the system.

22. The process according to any one of claims 1 to 21, characterized in that radical formers are added to the system.

23. The process according to any one of claims 1 to 22, characterized in that free-radical scavengers are added to the system.

24. The process according to any one of claims 1 to 23, characterized in that laccase, lignin peroxidase, manganese peroxidase or mixtures thereof are apportioned as enzymes. 95081 I,p:\oper'dab,17933.spe,16 17 Processes for the degradation of lignin, substantially as hereinbefore described with reference to the Examples. DATED this 14th day of August, 1995 Hans-Peter CALL By Its Patent Attorneys DAVIES COLLISON CAVE 950814,p:\oper\dab, 17933.spe, 17 18 ABSTRACT The present invention relates to a process for the degradation of lignin from material containing lignocellulose and/or bleaching of material containing lignocellulose for enzymatically deinking waste paper and for treating pulp and paper mill waste, whereby laccases obtained from Coriolus versicolor and other laccase formers are used by simultaneously adding and/or apportioning non-aromatic reducing and oxidizing agents as well as such phenolic compounds and/or non-phenolic aromatics which either oxidize the phenolic and non-phenolic lignin structures directly through the effect of these phenolic and/or non-phenolic aromatics or which oxidize the lignin through oxidation brought about by these compounds of further phenolic and/or non-phenolic compounds. INTERNATIONAL SEARCH REIPORT International application No. PCT/EP 92/01086 A. CLASSIFICATION OF SUBJECT MAT'ER Int.C1. 5 D21C5/00; D21C9/10; D21C5/02; C02F9/00 According to International Patent Classification (IPC) or to both national classification and IPC B. FIELDS SEARCHIED Minimum documentation searched (classification system followed by classification symbols) Int.C1. 5 D21C Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) C. DOCUMENTS CONSIDERED TO BE RELEVANT Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. P,X EP,A,0447673 (CALL,H. 25 September 1991 1-18,20, see the whole document 24 P,X EP,A,0429422 (ENSO-GUTZEIT OY) 1-2,5,6, 29 May 1991 11,12, 15,24 see page 2, line 24 line 34 Y PAPIER, DAS. 1-6 Vol.44,No. 10A, October 1990, DARMSTADT DE pages V33 V41 CALL, H.P. ET AL. 'Einsatz von lignolytischen Enzymen bei der Zellstoff- und Papierherstellung (Biopulping, Biobleaching).' see page V38, line 5 page Y WO,A,8803190 (CALL, 05 May 1988, 1-6 cited in the application see page 3, line 6 page 5, line 8 A 7-12,24 Further documents are listed in the continuation of Box C. See patent family annex. S Special categories of cied documents: later document published after the international filing date or prioritl document defining the general state of the artwhich is not considered dale and not in conflict with the application but cited to understand to be of particular relevance the principle or theory underlying the invention earlier document but published on or after the international filing date document of particular relevance: the claimed invention cannot be document which may throw doubts on priority claimn(s) or which is c dered v el r c e c onsi d e re d t o invole an inventive cited to establish the publication date of another citation or other stp when the document is taken alone special reason (as specified) document of particular relevance: the clailned invention cannot be document referring to an oral disclosure, use, exhibition or other considered to involve an inventive step when the document is means combined with one or more othersuch documents, such combination n" L .being obvious to a person skilled in the art document published prior to the international filing date but later than being obvious to a prson skilled in the a the priority date claimed document member of the same patent family Date of the actual completion of the international search Date of mailing of the international search report September 1992 (30.09.92) 20 October 1992 (20.10.92) Name and mailing address of the ISA/ Authorized officer EUROPEAN PATENT OFFICE Facsimile No. Telephone No. Form PCT/ISV/210 (second sheet) (July 1992) ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION No. EP SA 9201086 60247 This annex Limt the patent famnily members relating to the patent doc~umen~ts cited in the above-mentioned international search report. The members are as contained in the European Patent Office EDP file on The European Patent Office is in no way liable for these particulars which are merely given for the purpose of information. 30/09/92 Patent document Publication Patent famnily Publication cited in search report date member(s) I date EP-A-0447673 25-09-91 OE-A- 4008893 26-09-91 AU-A- 7543291 21-10-91 WO-A- 9114823 03-10-91 EP-A-0429422 29-05-91 CA-A- 2030186 18-05-91 JP-A- 3174078 29-07-91 WO-A-8803190 05-05-88 DE-A- 3636208 05-05-88 AU-B- 605215 10-01-91 AU-A- 8230387 25-05-88 EP-A- 0327576 16-08-89 JP-T- 2500990 05-04-90 SFor more details about this annex :see Official Journal of the European Patent Office, No. 12/82 INTERNATIONALER RECHERCHENBERIGHT PCT/EP 92/01086 lote-nitlonajes Aktenzeiche i. KLASSIFIKAION DES ANAEUJUNGSGEGENSTANDS (bel toehreren Klassifikatinssmbolen sind afle anzugebes)b Nach der Internacsonalen PatentIassifikation (IPC) oder nach der naitonalef KI2ssfitton und der IPC Int.K1 5 021CS/00; 021C9/10; D21C5/02; C02F9/00 UI. RECHiERCIERTE SACIIGEBIETE Reciserchierier Mind estprusioff hKassrfikationssylr K2sfkationssyenbole Int.K1. 5 ID21C Recherchierte nicht zurn Mindestprtifstoff geiibrende Ver~ffentlchungen, soweit diese unter die tedierchierien Sachgebiete fallen Mn. ELNSCI-LAGIGE VEROFFENTUCIILNGENI AM. kennzachnung der V'eroffentlichung 11, oweit e-fotderlich tinter Angabe der manigeblichen Teile 12 Stat. Anspruch N0~ 2 PX EP,A,0 447 673 (CALL, H. 1-18,20, September 1991 24 siehe das ganze Dokument PX EP,A,0 429 422 (ENSO-GUTZEIT QY) 1-3,5,6,

29. Mai 1991 11, 12, 15,24. siehe Seite 2, Zeile 24 Zeile 34 y PAPIER, DAS. 1-6 Bd. 44, Nr. 10A, Oktober 1990, DARMSTADT DE Seiten V33 V41 CALL, H.P. ET AL. 'Einsatz von lignolytischen Enzymen bei der Zelistoff- und Papierherstellung (Biopulping, Biobleaching) *1 siehe Seite V38, Zeile 5 -Seite 0 esondere K2tegorien von angegatbenen VarOffetstlichungen Verbffentlichung, die den aligesneinen Stad der Tedinik Sp~tere Vertiffentitc-hungR, die nach deen internationalen An- definiert, aber nicht 2us besonders bedeu tsarn anzusehen is meldedatumr oder dent Priorittsdatutn vettiffentlicht woiden E' Uteres Dokument, dan ledoch erst ame oder nads deen intet-na- tM tind mti der Anmeldung nicht kollidiert, sonde-ti our zuto tionalen Animeldedatuto vertiffenilicht wnrden ist Verst~ndnis des der Etfinioung zugrsindediegenden Pnnngs 'L Vertlentlichung, die geei gnet is, einen Priorittstsanspruch oder der ihr zugrstndeliegenden Theoie angegeben is zweifelhaft erscheinen zu lansess, oder dutch die dzs erof- Veroff ientllclsung von Isesonderer Bedeutung; die beatispruch- fentllichuinAtum einer anideren ito Reds eesbericht ge- te Et-findung satn nicht ams nest oder atol etiindensdser Ting- naninten Vertifentllchung beegt werien soil oder die ion einetnetbriedbercttwre 2aeten besnoderen Grund a.ngegeben is (wte a.usgefuhrt) Y' Verilifentlidnong von besonderei Bedeutung; die bezrispruch- 0' Verifentilcsu-ng, die sich aLf eine mUndliche Offesbarung, te Et-indn kann nidit ams auf erfindenschser Tatigkeit be- ruhend bes-chtet we-des, wenn die Verdrffentlidsong mit emne Benutzung, cane Ausnstellung oder anidere Mafloabmen einer odes menreren anderen Verhifentlir-sun gen dieser Kate- beniht got-ie in Verbindung gebracht stird tied diese Verbindung flit PF Verbffentlichung, die yor dent intetnationalen Atineldeda- esnen Fachenann naheliegend is tun, abet nach dent beanspruchren Prioritatsdanunt ve-fiffesut- W Verbiffentlic-hung, die Mieglied ders-elben Pateatfami-lie is Ilicht wordet s t 2 BESC}{ED IGUING Datum des Absclslusses der internationalen Rechetrche Absendedatum des intertsationalen kechecsesbe-sctts 1992

201.~ 92 lnte riauona e Recherchen kelorde Unt 11mch e de ns EUROPAISCRES PATENTANIT r FormUmt PCTjLSkj210 (BI-tt 2) jJ-w 1"51 yine Dagmar rIP~\Axu.i, LAMBfb.O Internaflonajles Aktenzeichen Ill. EINSCILAGIGE VERO FFENTJCI IUNG Ej (Fortsetzung von Blatt 2) PCT/EP 92/01086 Art 0Kennizelchnung der Veroffentichung, soweit erforderlich unter Ang-Ae der maflgeblichen Teie Betr. Ansi c r Y WO,A,8 803 190 (CALL, H. P.) S. Mai 1988 in der Anmeldung erwdhnt siehe Seite 3, Zeile 6 Seite 5, Zeile 8 A 1-6 7-12,24 ForTtIEL PCTIISAJZIO(Zo zk~.o~ezj (Jaow 19L51 IJM~ ~bJj~ F.mWNt PCTjIW2jo(Zuxzbcr"l (jagur 19s_41 Lj.%e MbLatt ANHANG ZUM INTERNATIONALEN RECHERCHENBERICHT OBER DIE INTERNATIONALE PATENTANMIELDUNG NR. EP 9201086 SA 60247 In diesczn Anhang sind dic Nlitgfieder der Patentfuruificn der im obengenannten internationadcn Recherchenberr~ht angefiihrten Patentdok-urnente angcgeben. Die Angaben Uber die Famniicnrnitglicdc.' entsprechen dem Stand der Datel des Eu-opAischen Patentaxnts urm Diesc Anigaben dienen nur zur Lnterricbtung und edfolgen ohne Gewiabr. 30/09/92 Im Recberchenbcrkbht Datum dr NMtglicd(cr) derDaudr an!efthrtcs Patcntdokurnnt Veroffentlichung PaetanleVerb ffentli ch ung EP-A-0447673 25-09-91 OE-A- 4008893 26-09-91 AU-A- 7543291 21-10-91 W0-k- 9114823 02-10-91 E P 0 4 2 9 4 2 2 29-05-91 CA--A- 2030186 18-05-91-- JP-A- 3174078 29-07-91 WO-A-8803190 05-05-88 DE-A- 3636208 05-05-88 AU-B- 605215 10-01-91 AU-A- 8230387 25-05-88 EP-A- 0327576 16-08-89 JP-T- 2500990 05-04-90 Fur nkhe Einztheiten zu diesern Anhang sice Arntsblatt des Europkischen Patentarnt, Nr.12/82