Indian Patents
Title of Invention

NOVEL LACCASE ENZYME
Abstract The present invention relates to a novel laccase enzyme obtainable from the strains of genus Thielavia.The invention relates also to the nucleic acid sequence encoding the enzyme, a recombinant host into which the nucleic acid sequence has been introduced and a method for the production of the enzyme in a recombinant host. The enzyme of the invention is suitable for several applications,in particular for increasing the lightness of denim.
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NOVEL LACCASE ENZYME AND USE THEREOF
FIELD OF THE INVENTION
This invention relates to a novel laccase enzyme useful in many applications, particularly in denim treatment This invention relates also to a nucleic acid encoding the enzyme, a vector, a host cell and a method for producing the enzyme as well as an enzyme preparation comprising the enzyme. Furthermore, this invention relates to methods for treating denim, methods for stain removal, methods for treating natural or man-made fibers or lignocellulosic fibres, methods for treating wool, methods for treating hair and methods for bleaching pulp and dye house effluents and methods for decolorizing dyes. This invention relates also to various uses and compositions, which can be used in the mentioned applications.
BACKGROUND OF THE INVENTION
Laccases (EC. 1.10.3.2 p-benzenediol: oxygen oxidoreductase) belong to a family of multi-copper oxidases. Laccases are widely distributed enzymes in higher plants, fungi, some insects and bacteria. They are characterized by low substrate specificity, oxidizing various substrates, including diphenols, polyphenols, different substituted phenols, diamines, aromatic amines, and even inorganic compounds like iodine. Laccases oxidize their substrates by a one-electron oxidation mechanism, and they use molecular oxygen as an electron acceptor. Among laccases the primary sequence, induction mechanism, physico-chemical (e.g. isoelectric point and carbohydrate content) and biochemical characteristics are variable. The copper binding sites of laccases are, however, strictly conserved.
Several laccase proteins and genes encoding these laccases have been previously isolated. WO 01/92498 describes a fungal laccase enzyme isolated from Melanocarpus albomyces strain. The enzyme having pH optimum within 5 to 8 and working at temperatures between 30 to 80°C is well suited to industrial applications-requiring-high-pH and-temperature conditions-whereas - the-majority-of-known fungal laccases function in an acidic pH range and are not very thermostable. The patent EP 0765394 Bl (corresponding U.S. Patent No. 5,981,243) describes the cloning of a laccase gene from Myceliopktkora thermophila and its expression in Aspergillus. The pH optimum of the laccase was 6.5 and the enzyme retained full activity 20 min in 60°C. U.S. Patent No. 5,750,388 describes the cloning of a laccase gene from Scytalidium thermophilum and its expression in Aspergillus. The pH profiles of Scytalidium laccase activity has optimal pH of 7 and 4 for syringaldazine and ABTS oxidation, respectively. The laccase was more thermostable at neutral to alkaline pH than at acidic pH.

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Laccases have many industrially potential applications, such as delignification of wood pulps, methods for treating lignin containing fibers, methods for treating wood fibers in order to functionalize them or glue the fibers, improval of the production of fuel ethanol from renewable raw materials, food applications (for example in baking or clarification of beer or wine), various bioremediative processes and textile applications, such as denim treatment, stain removal, treatment of various fibers for textile industry, methods for decolorizing dyes and methods for treating dye house effluents, or use in hair dyeing composition, in hard-surface cleaning or in detergent formulations.
"Stone washed" look or an abraded look has been denim producers' interest in recent years. Traditional stone washing with pumice stones reduces the strength of fabric and burdens the laundering apparatuses. Past years the trend has been towards enzymatic denim finishing processes. "Bleached look" of denim is normally obtained by means of sodium hypochlorite. So far this "chlorine bleaching" has been the most efficient bleaching method for denim dyed with Indigo, since almost all shades can be obtained. However, hypochlorite process is environmentally very harmful, it is difficult to control and it damages the fabric easily. It is also very inconvenient or even harmful method for the user, it cannot be used for Lycra containing products and antichlor treatment with several rinsing/washing steps is required. Intensive research has been underway for development of ecologically less harmful alternative for sodium hypochlorite, in particular laccases have been studied. So far the results with commercial laccase preparations cannot be compared to those obtained by "chlorine bleaching" as far as the effects and the looks are concerned- Heavy faded look has been very difficult or impossible to achieve without sodium hypochlorite.
WO 97/25468 describes the use of laccase in a mediod for providing to dyed denim an abraded look. The method comprises a cellulase treatment and simultaneous or subsequent treatment with a phenol oxidizing enzyme, such as laccase, and an enhancing agent, such as methylsyringate. Trametes villosa and Myceliophthora thermophila laccases are the examples of laccases in the patent publication.
Despite of the numerous publications describing laccases from various microorganisms, the prior art does not describe any.laccase which could be used jn_denim .treatment to replace the chemical bleaching-agents. There is thus a need for novel laccase enzymes having more efficient oxidizing capacity, in particular more efficient oxidizing capacity of Indigo.
There is also a need for novel laccases, which would function more effectively and be more suitable for the various conditions in different applications.

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SUMMARY OF THE INVENTION
It is an aim of the present invention to eliminate at least some of the problems associated with the prior art In particular, it is an aim of this invention to provide a novel laccase enzyme with which it is possible to decrease the amount or avoid the use of chemical bleaching agents in denim treatment. By using the laccase enzyme of the present invention, it is possible to decrease or avoid the use of chemical bleaching agents, such as sodium hypochlorite.
This invention is based on the surprising finding that laccase enzyme of the present invention can increase the lightness of denim as much as, or above, the level obtained by sodium hypochlorite. The laccase treatment increases the lightness of denim in particular on the face side of denim, which is also the desired result of denim treatment
One object of this invention is thus a laccase enzyme that under suitable conditions is capable of increasing the lightness of denim at least, or above, as many units as denim treated by sodium hypochlorite. The result obtained by laccase treatment of the present invention has been compared to bleaching result obtained with sodium hypochlorite at test conditions, which represent conventional sodium hypochlorite bleaching conditions.
More specifically, the laccase enzyme of this invention is characterized by what is stated in the characterizing part of claim 1.
This invention thus encompasses laccases, which are capable of increasing the lightness of denim on the face side of denim at least as many units as sodium hypochlorite under test conditions. Surprisingly, this effect can be achieved already when only desized denim is treated. When laccase treatment is made on desized and cellulase treated denim, the laccases of the present invention can increase lightness of denim at least 20 %, preferably at least 60 % more than sodium hypochlorite under test conditions.
One object of the present invention is a laccase enzyme that is capable, under suitable conditions, in a single treatment, to obtain lightness value (L*), on the face side of denim, at least 47, preferably at least 50, when Indigo dyed denim with lightness value (L*) on the face side of denim equal or lower than 32 after cellulase treatment is used for the laccase treatment
One object of the present invention is a laccase enzyme that is capable in a single treatment and under suitable conditions to obtain increase of lightness value (L*), on the face side of denim, at least 17 units, preferably at least 25 units, when Indigo dyed denim with lightness value (L*) on the face side of denim equal or lower than 32 after cellulase treatment is used for laccase treatment

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One object of the present invention is also a laccase enzyme, which comprises the amino acid sequence SEQ ID NO: 12 or a sequence showing at least 74 % identity to the sequence SEQ ID NO: 12 when the full-length sequences encoded by the corresponding genes are compared.
More specifically the laccase enzyme of this invention is characterized by what is stated in the characterizing part of claim 22.
The novel laccase of the present invention has high oxidizing activity towards various substrates. The enzyme is also capable of functioning in various conditions in different applications.
This invention encompasses enzymes, which have high oxidizing capacity of various substrates and are thus suitable for different applications. In particular, this invention relates to laccases having the specific activity of at least 800 nkat/mg on ABTS at pH 4.5 or at least 200 on syringaldazine at pH 5.5.
The enzyme is preferably obtainable from a microorganism, more preferably from a filamentous fungus, in particular from the genus Thielavia, more specifically from the species Thielavia arenaricu Advantageously, the enzyme is obtainable from the strain CBS 116071 deposited on 2 September 2004 at Centraalbureau voor Schimmelculmres, Upsalalaan 8,3584 CT, Utrecht, the Netherlands.
The laccase enzyme of the present invention functions at broad pH range. The enzyme functions ai pH 3.5 to 8, preferably at pH 4 to 7.5 under suitable conditions. Most preferably the enzyme functions at pH 5 to 7. The enzyme functions also at broad temperature range. For example, in denim treatment the enzyme is effective at the temperature of 30 to 80 °C, preferably at 50 to 70 °C, most preferably at 60 to 70 °C. The enzyme functions also at room temperature (18 to 30 °C), although with slower reaction rate than at higher temperatures.
One object of this invention is also a nucleic acid sequence encoding the enzyme of the present invention. The nucleic acid sequence is characterized by what is stated in the characterizing part of claim 44.
Further objects of this invention are a vector comprising thenucleic acid sequence of the invention and-a: host comprising the nucleic acid sequence or the vector, and a process for the production of a polypeptide having laccase activity.
One further object of the invention is a process for obtaining an enzyme preparation comprising the polypeptide or enzyme, which comprises the steps of culturing a host cell comprising the nucleic acid sequence encoding the enzyme of the invention or a vector comprising the nucleic acid sequence

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encoding the enzyme of the invention and recovering the polypeptide. Furthermore, an object of the invention is the enzyme preparation comprising the laccase enzyme of the invention.
One object of this invention is a method for treating denim, which comprises contacting denim in an aqueous medium with the laccase enzyme or enzyme preparation of the invention under suitable conditions for the function of the enzyme.
One object of this this invention is a method for removing stains, which comprises that material to be treated with the method is contacted with a laccase enzyme of the present invention under suitable conditions for the function of the enzyme.
This invention provides also a method of bleaching pulp, for treating fibers, a method for treating wool, a method for treating hair, a method for treating dye house effluents and a method for decolorizing dyes by using the laccase enzyme of the present invention.
Still further objects of this invention are uses of laccase enzyme of the present invention in various applications and compositions.
By using the laccase enzyme of this invention in denim bleaching it is possible to obtain many advantages. By using the laccase enzyme of this invention it is possible to decrease or folly avoid the environmentally harmful effects of sodium hypochlorite. If sodium hypochlorite is not used, no antichlor treatment is required. It is also possible to obtain various shades as by hypochlorite bleaching. Even a very faded look of denim can be obtained. One advantage of the laccase treatment by using the enzyme of the invention is that the treatment does not destroy the fabric. The laccase can also be used for treating Lycra containing products. In addition, the laccase treatment is also convenient for the user. Furthermore, the enzyme can function at a broad temperature and pH range. Also in other applications remarkable benefits can be obtained.
Other features, aspects and advantages of the present invention will become apparent from the following description and appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1. SDS-PAGE (15 %) showing the purification of Wiielavia laccase (TaLccl). Lanes: 1 MW marker (175, 83, 62, 47, 32.5, 25, 16.5, and 6.5 kDa), 2 culture supernatant, 3 fractions after DEAE Sepharose, 4-7 fractions after gel filtration, about 3-6 ug protein loaded on each lane. Proteins are stained with Coomassie Brilliant Blue.

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Fig. 2. pH optima of the purified Thielavia laccase (P TL) and the crude enzyme (CE) determined on guaiacol.
Fig. 3. Thermal stability of the wild type Thielavia laccase (TaL) at 50 and 60°C.
Fig. 4. Purification of the recombinant TaLccl, SDS-PAGE (12.5%). Lanes: 1 MW marker (175, 83, 62,47.5, 32.5, 25, 16.5 kDa) 2. culture supernatant, 3. fractions after DEAE Sepharose, 4. fractions after Resource Q 5-9. fractions after gel filtration.
Fig. 5. The tryptic peptide sequences obtained from the purified Thielavia arenaria wt TaLccl and the possible codons to encode the sequences.
Fig. 6 A and B. The nucleotide sequence of the Thielavia arenaria ALKO4197 Talccl gene and the deduced amino acid sequence. The stop codon is shown by an asterisk below the sequence. The location of the putative introns and the consensus intron splicing signals (5' GTPuNGPy, 3' PyAG, internal NNCTPuAPy) are marked by using lowercase letters and by holding, respectively. The putative signal peptide, analyzed by SignalP V2.0 program, and the mature C-terminal amino acid sequence (determined from the purified recombinant TaLccl protein) are underlined. The location of the tryptic peptide sequences obtained from the purified wt TaLccl are marked by dotted lines below the sequence. The conserved residues involved in copper binding are highlighted. The sites for putative N-glycosylation (N-X-S/T) are bolded.
Fig. 7. The expression cassette pALK.1667 used in the transformation of Trichoderma reesei protoplasts for producing the recombinant TaLccl. The laccase gene was under the control of the cbhl (cel7A) promoter (p cbhl) and termination of transcription was ensured by using the cbhl terminator sequence (t cbhl). The amdS gene was included as a transformation marker and the cbhl 3'-flanking region, together with the cbhl promoter, was used to enable targeting of the expression cassette into the cbhl locus by homologous recombination.
Fig. 8. The performance of TaLccl laccase preparation in denim bleaching at different pH values compared to DeniLite II Base. The enzyme dosage was 200 nkat/g and mediator dosage 10 mg/g on the weight of the fabric.
Fig. 9. Performance of TaLccl laccase preparation in denim bleaching at different temperatures compared to DeniLite II Base. The enzyme dosage was 200 nkat/g and mediator dosage 10 mg/g on the weight of the fabric.

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Fig. 10. The effect of enzyme dosage on bleaching of denim using TaLccl laccase preparation. The bleaching was performed as described in Example 9. DeniLite II Base was used for comparison.
Fig. 11 A - D. Bleaching of different types of denim with TaLccl laccase compared to bleaching with hypochlorite or DeniLite II Base. The bleaching was performed as described in Example 10. A. The results on bleaching Tincan Jeans, B. Lee Cooper Jeans, C. Warrick Jeans, D. English Jeans.
Fig. 12 A and B. Effect of TaLccl laccase preparation on grass soiling at 60°C compared to DeniLite II Base. The treatment was performed at pH 6 for 60 min. A. Lightness values, B. a* values (-a* is the green direction, +a* is the red direction).
Fig. 13 A and B. Effect of TaLccl laccase preparation on tea soiling at 60°C compared to DeniLite II Base. The treatment was performed at pH 6 for 60 min- A. Lightness values, B. a* values (-a* is the green direction, +a* is the red direction).
Fig. 14 A and B. Effect of TaLccl laccase preparation on grass soiling at 40°C with different dosages. The treatment was performed at pH 6 for 60 min. DeniLite II Base and mediator controls were used for comparison. A. Lightness values. B. a* values (-a* is the green direction, +a* is the red direction).
Fig. 15 A and B. Effect of TaLccl laccase preparation on tea soiling at 40°C with different dosages. The treatment was performed at pH 6 for 60 min. DeniLite II Base and mediator controls were used for comparison, A. Lightness values. B. a* values (-a* is the green direction, +a* is the red direction).
SEQUENCE LISTING
SEQ ID NO: 1 Sequence of the Peptide 1, a tryptic peptide from Thietavia arenaria ALKO4197 TaLccl protein.
SEQ ID NO: 2 Sequence of the Peptide 2, a tryptic peptide from Thielavia arenaria ALKO4197 TaLccl protein.
SEQ ID NO: 3 Sequence of the Peptide 3, a tryptic peptide from Thielavia arenaria-ALKO4197 TaLcccl protein.
SEQ ID NO: 4 The sequence of the oligonucleotide primer POX26. SEQ JD NO: 5 The sequence of the oligonucleotide primer POX27. SEQ ID NO: 6 The sequence of the oligonucleotide primer POX28. SEQ ID NO: 7 The sequence of the oligonucleotide primer POX29.

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SEQ ED NO: 8 The sequence of the oligormcleotide primer POX30.
SEQ ID NO: 9 The sequence of the oligonucleotide primer POX31.
SEQ ID NO: 10 The sequence of the PCR fragment obtained using the primers POX27 and POX31.
SEQ ID NO:11 The nucleotide sequence of the Thielavia arenaria ALKO4197 laccase 1 gene (Talccl).
SEQ ID NO: 12 The deduced amino acid sequence of the Tliielavia arenaria ALKO4197 laccase 1 (TaLccl).
Depositions
Tliielavia arenaria ALKO4197 was deposited at the Centralbureau Voor Schimmelcultures at Upsalalaan 8, 3584 CT, Utrecht, the Netherlands on 2 September 2004 and assigned accession number CBS 116071.
The E.coli strain RF5473 including the plasmid pALK1342 was deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Mascheroder Weg 1 b, D-38124 Braunschweig, Germany on 7 March 2003 and assigned accession number DSM 15484.
DETAILED DESCRIPTION
The present invention provides a laccase enzyme that is under suitable conditions, capable of increasing lightness of denim at least, or above, as many units as denim treated by chemical bleacing agent, in particular sodium hypochlorite.
By "the laccase of the present invention" or "the laccases of the present invention" is here meant the group of laccases as defined in the claims and described herein.
By "laccase enzyme" is in connection of this invention meant an enzyme classified as EC 1.10.3.2 by the enzyme classification. The laccase enzyme may originate from any organism including plants, preferably it may originate from microorganisms. It may originate from bacteria, for example from a genus selected from the group comprising Bacillus, Azospirillum and Streptomyces. Preferably the enzyme originates from fungi (including filamentous fungi and yeasts), for example from a genus selected from the group comprising Tfiielavia, Chaetomium, Achaetomium, Aspergillus, Botrytis, Collybia, Forties, Humicola,

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Hypocrea, Lentinus, Melanocarpus, Myceliophthora, Neurospora, Phlebia, Pleiirotus, Podospora, Polyporus, Rhizoctonia, Scytalidium, Pycnoponts, Trametes and Trichoderma.
According to a preferred embodiment of the invention the laccase of the present invention is obtainable from genus Thielavia, more preferably from Thielavia arenaria. According to a most preferred embodiment of the iaventioa the enzyme is obtainable from a strain deposited at Centraalbureau voor Schimmelcultures under number CBS 116071.
The origin of the laccase of the present invention is not restricted to genus Thielavia or to the species T. arenaria. By using the description provided herein, a person skilled in the art can find and isolate laccase of the present invention from other genera of fungi, from other microorganisms and also from higher organisms, such as plants.
Laccase of the present invention can be isolated from any organism producing laccase. Preferably the laccase enzyme of the present invention is isolated from a microbial source. Organisms capable of producing laccase can be screened, the activity on various substrates can be determined, and the enzyme characterized. For example, the pH and temperature ranges where the enzyme functions, pH and temperature optima, and enzyme stability at various temperatures, can be determined. Alternatively, genes encoding laccases in various organisms can be isolated and the amino acid sequences encoded by the genes can be compared with the amino acid sequence of the laccase isolated and characterized in the Examples here. This includes direct cloning from environmental samples.
"Bleached look" means the effects, which are obtained on denim fabric in the prior art by means of bleaching chemicals, e.g. sodium hypochJorite. So far the "chlorine bleaching" has been the most effective bleaching method for denim dyed with Indigo since almost all shades have been obtained with it If a "white bleaching" effect has been desirable, the bleaching has been carried out 2 to 3 times one after the other in different treatment baths, or by using high concentrations of hypochlorite. Bleaching with glucose, sulphinic acid derivatives or laccases have been suggested for denim treatment to replace sodium hypochlorite. However, in the prior art, wife none of them the same effects have been obtained as with hypochlorite.
To "increase the lightness" of denim fabric, according to the prior art, treatment with various bleaching chemicals or enzymes is carried out Bleaching is often done after treatment with cellulases or pumice stones, or both.
The "desizing" process is normally the first wet treatment of jeans and means the removal of starch or other sizing agents applied usually to the warp yams to prevent damage during the weaving process.

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Alpha-amylases are used to remove starch-based size for improved and uniform wet processing. After desizing the jeans are normally rinsed with water.
The term "abraded" means here the appearance of denim fabric when it has been treated by cellulase enzymes or stone washed, or both. As a result of uneven dye removal there are contrasts between dyed areas and areas from which dye has been removed. Synonymous expressions are "stone washed look" or "worn look". The cellulase treatment may be done using neutral or acid cellulases or both. If a fabric is not cellulase treated or stone washed, the appearance of the fabric is said to be "dull", since the fashionable contrasts would be missing.
By "increasing lightness" of denim is here meant a visible and measurable increase in the lightness of denim fabric. The increase of lightness is measured as lightness unit L* of the fabric after laccase treatment minus lightness unit L* before laccase treatment By "increasing lightness" of denim is meant in particular increasing lightness of denim on the face side of denim. The increase can be measured for example by measuring the colour as reflectance values with a spectrofotometer using L*a*b* color space coordinates as described in Examples 7-10.
The laccase enzyme of the present invention is capable, by single treatment, in suitable conditions, of increasing lightness of the face side of denim at least, or above, as many units as denim treated by sodium hypochlorite. The laccase treatment is made under conditions suitable for the function of the enzyme. The result of laccase treatment is compared with the result obtained with sodium hypochlorite under conventional bleaching conditions. As test conditions can be chosen the following bleaching conditions: fabric is treated in the presence of 25 ml/1 of 10% sodium hypochlorite (NaOCl) solution, at the temperature 40°C, at pH above 10.5, the treatment time being 15 min. The liquor ratio is about 1:15. The treatment can be carried out in equipments normally used for wet processes in textile industry, such as washing machines. The exact test conditions are described in Example 10. After bleaching the remaining hypochlorite is removed e.g. by treatment with sodium metabisulphite or sodium thiosulphate, followed by several rinsing/washing steps.
Compared to the result obtained under the mentioned sodium hypochlorite test conditions, the laccase enzyme of the present invention increases in suitable conditions, by single treatment, the lightness of desized denim of the face side of denim at least as many units as sodium hypochlorite. By "desized" is here meant "only desized", not cellulase treated. If laccase treatment is made on desized and cellulase treated denim, the increase of lightness is typically 20 to 70 %, preferably 40 to 115 % more than denim treated under test conditions by sodium hypochlorite.

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With the laccase of the present invention it is possible, in a single treatment and under suitable conditions, to obtain lightness value (L*), on the face side of denim, from 47 to 50 or even higher, when Indigo dyed, cellulase treated denim with lightness value (L*) on the face side of denim lower than 32 is used.
With the laccase of the present invention it is possible, in a single treatment and under suitable conditions, to obtain increase of lightness value (L*), on the face side of denim, from 17 to 25 units or even higher, when Indigo dyed, cellulase treated denim with lightness value (L*) on the face side of denim lower than 32 is used.
By treating denim fabric with the laccase of the present invention it is possible to obtain a bleaching effect akin to what is obtained with sodium hypochlorite. If more whitish effect is desired, higher dosages can be used or the enzyme treatment can be repeated or combined with other bleaching methods. The laccase treatment of the present invention can be combined also with any other bleaching treatment, with one or more chemical bleaching treatments or with one or more other enzyme treatments having capability of increasing lightness of denim.
By "mediators" are here meant additives, which are often needed for enhancing the effect of laccases. Many of the prior art laccases do not function or do not function effectively in the absence of mediators. Also the laccase originating here from Thielavia functions more effectively in the presence of mediators. Suitable mediators include, for example methyisyringate, acetosyringon, ethylsyringate, butylsyringate and laurylsyringate, propionic acid-phenothiazine (PPT) 2,2'azinobis-3-ethylbenztbiazole-6-sulphonate (ABTS), 2,2,6,6-tetramethyl-l-piperidinyloxy (Tempo), 1-hydroxybenzotriazole (HBT), violuric acid, N-hydroxy-acetanilide (NHA). The mediator may be used in the range 0.1 to 100 mg/g or 0.1 to 100 mg/1, preferably 1 to 10 mg/g or 1 to 10 mg/1 of the treated material depending on the application and on the mediator.
With the laccase enzyme of the present invention any kind of denim fabric can be treated. Advantageously the denim is Indigo dyed denim. Denim can also be treated with derivatives of Indigo or denim dyed with Indigo together with some other dye, for example indigo-dyed denim with sulphur bottom. The denim fabric may be cellulase treated or stone washed, or both, or the denim fabric may be treated by laccase of the present invention already after desizing.
According to a preferred embodiment of this invention denim treatment by the laccase of the present invention is carried out at the temperature of 30 to 80°C, preferably at the temperature of 50 to 70 °C, more preferably at the temperature of 60 to 70°C. The pH during the treatment may be in the range from pH 3.5 to 8, preferably from pH 4 to 7.5, most preferably from pH 5 to 7. The treatment may be carried

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out in 15 minutes to 2 hours, preferably in 30 minutes to 90 minutes, more preferably in 30 minutes to 60 minutes. The dosage used in the treatment can be 2 to 500 nkat, more preferably 20 to 200, most preferably 20 to 100 nkat/ g fabric.
The denim treatment according to the invention comprises generally the following steps:
desized or optionally desized and eellulase treated denim is contacted in aqueous medium with
an effective amount of laccase enzyme under suitable conditions for the function of the enzyme;
and
one or more rinses with water are carried out
The laccase treatment is preferably carried out on cellulase treated denim. Laccase treatment is followed by one or more rinses with hot or cold water optionally with detergents. Enzyme inactivation is usually not needed after laccase treatment since it does not reduce the strength of fabric, but if needed can be carried out by methods well known to a person skilled in the art The treatment is typically carried out in an equipment normally used for wet processes in textile industry, such as industrial machines used for washing, cellulase treatment, dyeing or finishing.
By "denim" is in connection of this invention meant denim fabric, usually denim jeans.
Performance of the laccase preparation of the present invention in denim bleaching was exemplified at different pH-values as described in the Example 7. Recombinant laccase preparation produced using TJtrichoderma as a host was tested for its ability to bleach denim and compared to a commercial laccase preparation DeniLite II Base from Novozymes.
The laccase of the present invention was superior in decolorisation of indigo dye of denim compared to the prior art laccase at all pH values from 5 to 7, pH 6 being the optimum as can be seen in Table 10 and in Figure 8. Only the laccase preparation of the present invention was capable of achieving a strongly bleached look with the highest lightness value. In addition, the abraded look obtained by cellulase treatment was maintained. The increase of lightness and the decrease of blueness on the reverse side of denim was also the highest with denim treated with the laccase preparation of the invention with the mediator.
The ability of the laccase of the present invention to bleach denim at different temperatures was tested and compared to the prior art laccase as described in Example 8.
The laccase preparation of the present invention was superior in bleaching of denim (higher increase of lightness) compared to the prior art laccase at 30 to 80 °C as can be seen in Table 12 and in Figure 9.

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The temperature 60 to 70 °C was the most optimal for the laecase of the present invention and the look of the denim fabric was strongly faded.
Effect of enzyme dosage on bleaching of denim was studied with laccase-mediator system as is described in Example 9. Cellulase treated denim jeans were treated with recombinant laccase product from Trichoderma strain and the prior art iaccase using different iaccase dosages. The increase of the dosage from 20 to 100 nkat/g of fabric greatly improved the bleaching with the enzyme of the present invention. Increasing the time from 30 min to 60 min further improved the performance of the Iaccase of the present invention as can be seen in Table 14 and in Figure 10.
Bleaching of denim with laccase-mediator system was compared to bleaching with hypochlorite as is described in Example 10. Different types of denim were treated with the Iaccase of the present invention and the prior art Iaccase and the results obtained were compared to hypochlorite bleaching. Denim samples washed with cellulase after desiring to different abrasion levels or only desized samples were used in the test The Iaccase of the present invention was superior compared to the prior art Iaccase and chlorine bleaching with all cellulase treated denim samples of each type as can be seen in Tables 18-20 and in Figures 11 A to D. With fabrics that had only been desized the bleaching effect (increase of L* on the face side of denim) obtained with Thielavia Iaccase preparation was equal or better than with the sodium hypochlorite and at least 100 % better than with the prior art Iaccase (Denilite II Base). With the iaccase of the present invention it was possible to achieve a very strong bleaching effect that is normally obtained only with the use of high amounts of sodium hypochlorite.
Microorganisms that produce the Iaccase of the present invention can be isolated from nature or they can be screened from already isolated and identified strains of culture collections by using screening methods that are well known for a person skilled in the art Screening can be carried out by studying the production of the enzyme either on a solid culture on plate cultivations or in a liquid culture medium by measuring the enzyme activity. Suitable substrates for measuring the activity include ABTS, di-methoxyphenol (DMP), guaiacol, and syringaldazine. Fungi can be screened for their ability to produce laccases for example by the methods referred in Example 1 with indicators, such as Remazol Brilliant Blue R-478, tannic acid, and guaiacol. Suitable laccases can be isolated and the genes encoding them can be cloned aiso from higher organisms, such as plants.
This invention relates to enzymes which have high oxidizing capacity of various substrates and is thus suitable for different applications. In particular, this invention relates to enzymes having the specific activity of at least 800 nkat /mg, more preferably at least 900 nkat/mg on ABTS at pH 4.5. When syringaldazine is used as subtrate the specific activity is at least 200, preferably at least 300 nkat /mg at

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pH 5.5. When guaiacol is used as substrate the specific activity of the enzyme is at least 40 nkat/mg, preferably at least 60 nkat/mg at pH 5.5.
Microorganism strains, which are found as a result of screening can be cultivated on a suitable medium, and the formation of laccase in the culture solution or plate can be observed. After a sufficient amount of an interesting iaccase has been produced, the enzyme can be purified and its properties can be more thoroughly characterized.
The produced laccase enzymes can be purified by using conventional methods of enzyme chemistry, such as salt precipitation, ultrafiltration, ion exchange chromatography, and hydrophobic interaction chromatography. Purification can be monitored by protein determination, enzyme activity assays and by SDS polyacrylamide gel electrophoresis. The enzyme activity of the purified enzyme at various temperatures and pH values can be determined; similarly, the molecular weight and the isoelectric point can be determined.
The purified enzyme refers to an enzyme preparation, which has no other proteins or very low amount of other proteins in addition to the laccase protein. The purity of the obtained laccase that is essentially free from other proteins is >90%.
The purification of a preferred laccase of the present invention has been exemplified in Example 1. Concentrated culture filtrate was loaded on Q Sepharose FF column, proteins were eluted with an increasing salt gradient and laccase active fractions were loaded on Sephacryi S100 gel filtration resin. Purification was followed by activity assays and by SDS-PAGE. In order to obtain high purity samples an additional Resource Q anion exchange step was included. Naturally, it is possible to separate the enzyme of the present invention by using other known purification methods instead, or in addition to the methods described here.
The isoelectric point of the laccase can be determined with isoelectric focusing and bands containing laccase activity can be visualized by staining the gel with ABTS, for example, as described in Example 2.
Determination of laccase activity at various temperatures can be carried out by the ABTS method, as described in Example 1 in accordance with the method developed by Niku-Paavola et al. (1988) or by other methods described in literature.
The pH optimum of the laccase can be determined in a suitable substrate in a suitable buffer at different pH values by following the activity.

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The thermal stability can be determined by incubating an enzyme sample at various temperatures in a suitable buffer at a certain pH. The residual activity of the enzyme at each temperature can be defined by the ABTS method, for example.
Specific activities of the purified laccase can be determined towards different laccase substrates, such as ABTS, di-metoxy-phenol (DMP), syringaldazine, and guaiacol.
The effect of various inhibitors on laccase activity can be determined by measuring the oxygen consumption during the enzyme reaction with ABTS, for example, in sealed and filled containers with oxygen electrode or following the enzyme activity by spectroscopic means in the presence of an inhibitor.
The N-terminus of the protein as well as the internal peptides can be sequenced according to Edman degradation chemistry [Edman P. and Begg G. (1967)] as described in Example 2 or by other methods described in the literature.
The molecular weight of the purified laccase enzyme isolated from Thielavia arenaria RF5597 was approximately 80 kDa. The purified laccase showed multiple bands in isoelectric focusing at pis 5.5, 5.9, 6.4, 6.8, and 6.9.
The pH optimum for the purified laccase was 5.5, determined on guaiacol, and the enzyme showed substantially high activity still at pH 7. The enzyme functions at pH range from 3.5 to 8, preferably from 4 to 7.5 and most preferably at 5 to 7. The accurary of the measurement is ± 0.5.
The first pH range (pH 3.5 to 8) means that 20 % or more of the maximal activity is within this range, the second pH range (pH 4 to 7) means that 40 % or more of the acitivity is within this range. The third region (pH 5 to 7) means that 80 % or more of the activity is within this range.
The half life of the laccase was 26 br at 50 °C and 5.5 at 60 °C in the assay conditions.
The temperature optimum of the laccase in the denim bleaching application was 60 'C. The enzyme functions at the temperatures 18 to 80 "C, although effectively at temperatures 30 to 80 *C, more preferably at temperatures 50 to 70 °C, most preferably at the temperatures 60 to 70 "C.
The specific activity of the enzyme was the highest on ABTS, 1020 nkat/mg of protein at pH 4.5. The specific acitivity on DMS was 260, on syringaldazin 490 and on guaiacol 63 nkat/mg at pH 5.5.
The laccase which shows advantageous properties may be either produced by the original or recombinant host by a method comprising cultivating under suitable conditions a host into which a DNA sequence

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encoding said laccase and sequences needed for expressing said enzyme, has been introduced, and optionally isolating the enzyme. The production host can be any organism capable of expressing the laccase. Preferably the host is a microbial cell, more preferably a fungus. Most preferably the host is a filamentous fungus. Preferably the recombinant host is modified to express and secrete laccase as its main activity or one of its main activities. The spent culture medium of the production host can be used as such, or the host cells may be removed, and/or it may be concentrated, filtrated or fractionated. It may also be dried.
Suitable expression and production host systems are for example the production system developed for the fungus host Trichoderma (EP 244 234), or Aspergillus production system, such as A. oryzae or A. niger (WO 9708325 and WO 9533386, US 5,843,745, US 5,770,418), or the production system developed for Fusarium, such as F. oxysporum (Malardier et al., 1989) or for Chrysosporium (US 6,573,086). Suitable production systems developed for bacteria are a production system developed for Bacillus, for example B. subtilis or for E. coli, or for actinomycete Streptomyces. Suitable production systems developed for yeasts are systems developed for Saccharomyces, Shizosaccharomyces or Pichia pastoris. Production systems in some other microbes or in mammalian cell are also possible.
Preferred hosts for producing laccase enzyme of the present invention are in particular strains from genus Trichoderma or Aspergillus.
Within the scope of protection are also vectors which can be used when the nucleic acid sequence encoding the chosen laccase are introduced into a host and sequences facilitating the expression and secretion of the laccase encoding sequence, such as promoters and signal sequences.
Standard molecular biology methods can be used in the cloning of the laccase enzyme i.e. in the isolation and enzyme treatments of DNA, in E. coli transformations, etc. The basic methods used are described in the standard molecular biology handbooks, e.g. Sambrook et al. (1989) and Sambrook and Russell (2001).
Genomic library prepared from the chosen host .organism was screened with probes prepared by PCR. The sequences of the oligonucleotide primers used in the PCR reactions based on the amino acid sequences of the peptides obtained from the purified laccase enzyme produced by the natural host and on the concensus sequences of fungal laccases. The DNA products obtained were characterized by sequencing and by performing Southern blot hybridizations to the genomic Thielavia DNA digested with several restriction enzymes.

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The full-length Thielavia laccase gene was included in the plasmids pALK1607, pALKI341 and pALK1342. The Exoli strain RF5473 including the plasmid pALK1342 was deposited to the DSMZ collection (DSM 15484). The deduced amino acid sequence of the laccase was analyzed from the DNA sequence.
The Thielavia laccase Talccl sequence (SEQ ID NO: 11) and the deduced amino acid sequence (SEQ ID NO: 12) are shown in Fig. 6. The length of the gene was 2279 bp (including the stop codon). The deduced protein sequence consisted of 617 amino acids including a predicted signal sequence of 21 amino acids and a "tail" sequence of 13 amino acids (starting after the sequence DSGL). The peptides purified from the wild type laccase were ail found from the deduced amino acid sequence indicating that the gene cloned encodes the laccase purified from the recombinant host The predicted molecular mass was 64456 Da for the mature polypeptide and the predicted pi was 6.31 (amino acids 22 - 604, signal sequence and the C-terminal tail removed). The deduced amino acid sequence included nine putative N-glycosylation sites. The homologies to the published laccase sequences were searched using the BLAST program, version 2.2.9 atNCBI (National Center for Biotechnology Information) (Altschul et al., 1990). The highest homologies were found to the laccases from Melanocarpus albomyces, Podospora anserina and Neurospora crassa (EMBL accession numbers CAE00180.1, LAC2_PODAN, XP_323881.1). The TaLccl sequence was aligned with the sequences with identity above 50% in the BLAST search and with the laccase sequences from Myceliophthora thermophila (EP 0765394 Bl, corresponding U.S. Patent No. 5,981,243) and Scytalidium thermophilum (U.S. Patent No. 5,570,388). The highest identity, 73,1%, was found with Melanocarpus albomyces laccase.
By the term "identity" is here meant the identity between two amino acid sequences compared to each other from the first amino acid encoded by the corresponding gene to the last amino acid. The identity of the full-length sequences is measured by using Needleman-Wunsch global alignment program at EMBOSS (European Molecular Biology Open Software Suite; Rice et al., 2000) program package, version 2.9.0, with the following parameters: EMBLOSUM62, Gap penalty 10.0, Extend penalty 0.5.
Within the scope of the present invention are enzymes or poLypeptides which comprise amine-acid sequences which have laccase activity and which show at least 74 % identity to the amino acid sequence SEQ ID NO: 12. Preferred enzymes comprise amino acid sequences which show at ieast 76 %, more preferably at least 78 %, even more preferably at least 80 % identity. Still more preferable the amino acid sequences show at least 85%, more preferably at least 90 %, most preferably at least 95 % identity to the amino acid sequence SEQ ID NO: 12.

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Within the scope of the present invention are also enzymes and truncated polypeptides as defined above, but which lack signal sequence or tail or both. The signal sequence or the tail or both may be cut for example during posttranslational phases of the production or in the spent culture medium or during the storage of the culture medium or enzyme preparation. En addition, a propeptide from the protein may be cleaved by the host The truncation can also be achieved e.g. by shortening the gene encoding the polypeptide prior to transforming it to the production host.
The laccase according to the invention can be produced to the culture medium of its natural host or a recombinant host, from where it can be isolated and purified by using known methods of protein chemistry. If the culture medium contains a sufficiently high amount of laccase but no other detrimental proteins, it is possible to use the culture solution as such by simply separating the cells. When so desired, the culture solution can be concentrated, filtrated, fractionated and/or purified. It may also be dried. It is preferable to use, in various applications, an enzyme preparation containing an increased amount of laccase. Such an enzyme preparation can be prepared by producing the increased amount of laccase enzyme in the culture medium of the production host by means of gene technology or by optimising the cultivation conditions. The increased amount refers to an amount of laccase enzyme, which exceeds the amount of laccase enzyme naturally produced by the natural host.
According to a preferred embodiment of the invention Thielavia laccase can be produced in a filamentous fungus host, preferably in a Trichoderma host The production is described in more detail in Example 4. The purified recombinant laccase was characterized in terms of pH optimum, thermal stability, and pi, which clearly indicated that the recombinant laccase has similar properties as the wild type Thielavia laccase.
The production of laccase can also be improved by optimising the culture conditions and the culture medium of a wild or a recombinant strain. The carbon/nitrogen ratio can be optimised to be the best for the production of enzyme. The growing conditions, pH, temperature, mixing and air supply can be optimised to be the best possible for the enzyme production in question. In fermentation, inducers of laccase production, such as veratryl alcohol, xylidine,.or lignin car also be used.The wayand the time of adding the inducers, as well as their concentration can be optimised.
The term "enzyme preparation" denotes here to any enzyme product, which contains at least one laccase enzyme. Thus, such an enzyme preparation may be a spent culture medium or filtrate containing one or more laccases or one or more laccases and other enzymes, an isolated laccase enzyme or a mixture of one or more laccase enzymes or a mixture of one or more laccase enzymes and one or more other enzymes. In addition to the laccase activity, such a preparation may contain additives, such as mediators, stabilizers, buffers, preservatives, surfactants and/or culture medium components. Preferred additives are

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such, which are commonly used in enzyme preparations intended for the application, where the enzyme preparation is used. The enzyme preparation may be in the form of liquid, powder or granulate.
By "spent culture medium" is here meant the culture medium of the host comprising the produced enzymes. Preferably the host cells are separated from the said medium after the production.
The enzyme preparation may comprise in addition to laccase, one or more other enzymes, which may be for example amylases, cellulases and/or peroxidases. Alternatively, before, during or after the laccase treatment of the present invention, another enzyme treatment may be carried out The enzyme treatment may comprise, for example, one or more amylase treatments, one or more cellulase treatments and/or one or more peroxidase treatments. Which other enzymes are included to the enzyme preparation or are used in the enzyme treatment, depends on the application.
The enzyme preparation may comprise one or more laccase enzymes of the present invention or other laccase enzymes together with one or more laccase enzymes of the present invention. For example, laccase enzymes having different properties may be combined to make the enzyme preparation more useful for different conditions.
The laccase enzyme of the present invention can be used in stain removal under similar conditions as in denim bleaching.
According to a preferred embodiment of this invention stain removal is carried out at the temperature of 30 to 80°C, preferably at the temperature of 50 to 70 °C, more preferably at the temperature of 60 to 70°C. The pH during the treatment can be from pH 3.5 to 8, preferably from pH 4 to 7.5, most preferably from pH 5 to 7. The treatment may be carried out in 15 minutes to 2 hours, preferably in 30 minutes to 90 minutes, more preferably in 30 minutes to 60 minutes. The dosage used in the treatment can be 0.2 to 2000 nkat/g, preferably 1 to 500 nkat/g, more preferably from 2 to 200 nkat/g on the weight of fabric.
The laccase of the present invention and DeniLite II Base laccase preparations were tested for their ability to remove stains as is described in Example 11. In the tests artificially soiled test cloths for grass soiling and for tea-soiling were used with or without the mediator. The dosages of the enzyme were 20 and 200 nkat/ g of fabric and the test was run at 40, 50 or 60°C and pH 6 for 60 min.
The laccase preparation of the present invention was more effective in removal of grass soiling with the mediator at 60°C compared to the prior art laccase as can be seen in Table 21 and in Figures 12 A and B. The laccase of the present invention was also better at 40°C as can be seen in Table 22 and in Figures 14 A and B. Mediator was needed to obtain the desired effect

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The laccase of the present invention was also the more efficient in removal of tea stain with the mediator at 60°C and slightly better even at 40 °C especially with higher dosage as can be seen in Tables 21 and 22 and in Figures 13 A and B and in Figures 15 A and B. Mediator was needed to obtain the desired effect.
The laccase of the present invention was also more efficient in removal of tea stain with the mediator at 60°C and slightly better even at 40 °C especially with higher dosage as can be seen in Table 22 and in Figure 14. Mediator was needed to obtain the desired effect.
The laccase enzyme of the present invention can be used also in decolorization of dyes. Dye-house effluents, for example cannot be discharged to natural waters without removing the dyes and/or decolorizing them. The decolorization can be carried out under similar conditions as used in denim bleaching. Suitable dosage of the enzyme and treatment time depends on the amount of the dye to be decolorized and the treatment conditions.
According to a preferred embodiment of this invention decolorization of dyes is carried out at the temperature of 30 to 80°C, preferably at the temperature of 50 to 70 °C, more preferably at the temperature of 60 to 70°C. The pH during the treatment can be from pH 3.5 to 8, preferably from pH 4 to 7.5, most preferably from 5 to 7. The enzyme dosages and treatment times can be tested and chosen to be most suitable for the application. As guidance the dosages of 0.2 to 2000 nkat/1 of the treatment solution can be used. The treatment time is preferably 15 min to 24 hours, more preferably 30 minutes to 12 hours. If the treatment is carried out at lower temperature, for example 18 to 30 °C the treatment time may be longer.
As described in Example 12 the laccase of the present invention was tested for its ability to decolourize different dyes in the presence or absence of a mediator. The shake flasks were incubated at 50°C for ca. 30, 90 and 150 minutes. The laccase was able to decolourize Indigocarmine, Remazol Brilliant Blue and Cibacron Brilliant Red 3B-P to great extend as can be seen in Table 23.
Since the laccase of the present invention have high oxidizing capacity, of various substrates^ it is well suited for many industrial applications. Such applications are for example the manufacture of fibre products and applications of forest industry, applications in the cosmetic industry and in the industry preparing personal care and other applications. In these applications, the temperature and pH are on the area where the laccase of the present invention function. The dosage and treatment time can be chosen depending on the application and material to be treated.

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Mediators may be needed as additives to enhance the effect of the laccases of the present invention. In addition, it is essential that enough oxygen is brought to the reaction. If needed, oxygen can be added either by bringing air or oxygen or air enriched with oxygen to the reaction mixture.
The laccase of the present invention is suitable for use in the textile industry, for treating man-made or natural fibers or their combinations. The enzyme is suitable for treating cellulosic fibers as well as proteinaceous fibers, such as wool.
The laccase of the present invention is suitable for use in the forest industry. Lignin-containing fibres can be brought into contact with the laccase. Due to the laccase treatment, the strength properties of the fibres improve, which can be utilised, for example, in the manufacture of fibre boards, in wood composites, in paper or cardboard product and composites, which are made of mechanically ground lignin-containing fibres. Wood fibers can be treated with laccases of the present invention also to functionize them or glue the fibers.
The laccase of the present invention is also well suited to depolymerization of various compounds. By using the laccase of the present invention lignin in kraft pulp can be depolymerised thereby producing a pulp with lower lignin content Laccase can thus be used for bleaching of pulp to decrease the use of bleaching chemicals. As a result of the better bieachability of the pulp after laccase treatment, there is a reduction of the subsequent consumption of bleaching chemicals, which when chlorine containing chemicals are used, leads to a reduced formation of environmentally undesired organo-chlorinc compounds.
The laccase of the present invention can be used also for polymering compounds, such as lignin, to produce high molecular weight compounds.
Because of the high oxidizing capacity of the enzyme it can be used for oxidizing of dyes or dye precursors or chromophoric compounds in cosmetic industry or in industry preparing products for personal care. The oxidation of the dyes leads to decolorization of the compounds. This effect can be used for example in hair dyeing or when whitening teeth. To carry out hair dyeing dye precursors or modifiers are usually needed.
The laccase according to the invention can also be used to improve the runnability of paper machines. The laccase can be used to improve the runnability of paper machines by polymerising compounds originating from lignin and extractives and by decreasing the detrimental growth of microbes in the paper machine.

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Further possible applications where laccase enzymes of the present invention can be used are methods for improving doughs in baking applications, methods for clarifying beer and wine, use in improval of the production of fuel ethanol from renewable raw materials and use in various bioremediative processes as well as use in hard-surface cleaning or in detergent formulations.
In general, in the mentioned applications the treatment temperature is preferably 30 to 80 °C, more preferably 50 to 70 °C, although reactions can be carried out also in lower temperatures, such as at temperatures 18 to 30 °C. The pH may be 3.5 to 8, preferably 5 to 7. The treatment time may be 15 min to 24 hours, preferably 30 min to 2 hours. The dosage may be 0.1 to 2000, preferably 1 to 1000, more preferably 2 to 200 nkat/ g or I of the material to be treated. A suitable amount of a suitable mediator may be added.
Compositions for the mentioned applications comprise the enzyme or enzyme preparation of the present invention in an effective amount and optionally additives suitable for the application in question. Compositions for textile industry may comprise for example a suitable amount of surface active agents, buffers stabilizers and preservatives, compositions for forest industry may comprise for example a suitable amount of buffers, stabilizers and preservatives. In all compositions should be avoided substances harmful for environment and for human (or animal) use. In particular compositions for cosmetic industry and industry for personal care products should not contain harmful effects on skin or as ingested.
The present invention provides composition for the treatment of denim comprising a laccase enzyme or an enzyme preparation according to the invention. The present invention provides also a composition for the removal of stain, a composition for the bleaching of pulp, a composition for the treating of fibre for textile industry, a composition for the treating of fibre for forest industry, a composition for the treating of wool, a composition for the treating of hair, a composition for the treating of dye house effluent, and a composition for the decolorizing of dyes comprising a laccase enzyme or an enzyme preparation according to the invention.
The following examples are intended for illustration of the present invention and should not be interpreted as limiting the present invention in any way.

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EXAMPLE 1. Production and purification of the Thielavia laccase
Production of the Thielavia laccase
Various strains from the culture collection of Roal Oy were screened for their ability to produce Iaccases with indicators Remazo! Brilliant Blue R-478, tannic acid, and guaiacol as described in Kiiskinen et aL (2004). Thielavia arenaria ALKO4197 showed positive reactions on guaiacol and Remazol Brilliant Blue R-478.
Thielavia fungus was maintained on PD agar (Difco) at +4°C. The inoculation and production medium contained: 25 g/1 glucose (AnalaR), 27.5 g/1 Bacto yeast extract (Difco), 0.5 mg/ml Indulin AT (Sigma), 0.04 1/1 mineral solution (1.0 g/1 CaCl2 • 2H2O (Riedel-de Haen), 1.0 g/1 FeSO4 • 7H2O (Riedel-de Haen), 0.1 g/1 ZnSO4 • 7H2O (Merck), 0.16 g/1 CuSO4 • 5H2O (Merck), 1.0 g/1 Na2EDTA (Riedel-de Haen)). Glucose was sterilized separately and combined aseptically to the medium.
The microbe was cultivated in 50 or 200 ml volume on a rotary shaker (200 rpm) at temperature of 37°C. The medium was inoculated with 5 or 20 ml of well-grown mycelia. The laccase activity was followed up to eight days and the highest laccase activity (about 20 nkat/ml) was reached after six days of cultivation. Six parallel cultivations were made. Cells were removed from the fermentation broth by centrifugation (10 000 g for 10 min, at +4°C) and the culture filtrate was further purified
Purification of the Thielavia laccase
Concentrated culture filtrate was first loaded on Q Sepharose FF column, which was pre equilibrated with 10 mM Tris HCL, pH 8.5. Proteins were eluted with an increasing salt gradient (0-500 mM Na2SO4 in the equilibrating buffer). Laccase active fractions were pooled and loaded on Sephacryl SI00 gel filtration resin, which was equilibrated with 20 mM Tris-buffer, pH 7.0, containing 200 mM NaCl. Purification was followed by SDS-PAGE stained with Coomassie brilliant Blue. Laccase positive fractions were pooled and concentrated. Salts were removed and buffer changed to 20 mM Tris buffer, pH 7:0. In order ~1o obtain high purity samples an additional Resource Q anion exchange step was included. The sample was loaded onto a Resource Q column, which was equilibrated with 10 mM Tris HC1 pH 8.5. Proteins were eluted with a linear 1-300 mM Na2SO4 salt gradient Fig.1. shows the purification of the laccase.

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Enzyme activity assay
The laccase activity from the culture supernatant was measured using ABTS as substrate. The activity assay was carried out in accordance with the method developed by Niku-Paavola et al. (1988). The sample was diluted with 0.025 M succinate buffer, pH 4.5. First 0.350 ml of ABTS solution (11 g/1) was added to 1.15 ml of the dilution, and the reaction was followed for 2 minutes at a wavelength of 436 nm. The activity is expressed as nano katals.
Determination of protein contents
The protein contents were determined by the DC Protein Assay kit of Bio-Rad, based on a method developed by Lowry et al. (1951). The assays were carried out according to the supplier's instructions, and the intensity of the colour formed in the reaction was measured on a wavelength of 750 nm using the Perkin Elmer Lambda 20 spectrophotometer. A standard curve was defined using bovine serum albumin in concentrations of 0.25-1.25 g/1 (BSA, Bio-Rad).
EXAMPLE 2. Characterization of the purified Thielavia arenaria laccase, wt TaLccl
Molecular weight and pi
Molecular weight of the Thielavia arenaria laccase (wt TaLccl) was determined on SDS-PAGE according to Laemmli (1970). The gels used in the SDS-PAGE analysis were ready-made 12% Tris HC1 gels (BioRad). Protein bands were visualized by staining with Coomassie Brilliant Blue (R 350; Pharmacia) and compared with molecular weight markers (Prestained Protein Marker Broad Range #7708S; New England BioLabs, Beverly, Mass.). The molecular weight of the Thielavia areanaria laccase was approximately 80 kDa. The isoelectric point of the laccase was determined with isoelectric focusing within the pH range of 3-9 (Pharmalyte IEF, Pharmacia) on a LKB 2117 Multiphor II Electrophoresis System (LKB Pharmacia, Bromma, Sweden) according to the manufacturer's instructions. Bands containing laccase activity were visualized by staining the gel with 2 mM ABTS in 25 mM succinate buffer (pH 475) and proteins by Coomassie Blue staining. The purified Thielavia laccase showed multiple bands in isoelectric focusing at pis 5.5, 5,9, 6,4, 6.8, and 6.9.
pH optimum
The pH-optimum of the TJiielavia arenaria laccase was determined in the universal McIIvaine buffer within a pH range of 2.0-8.0 using guaiacol as substrate. The pH optima determined for the purified and

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crude Thielavia laccase are shown in Fig. 2. As shown in Fig. 2 the pH optimum for Thielavia Iaccase is at 5.5, the enzyme shows substantially high activity still at pH 7, above which the activity starts to drop.
Thermal stability
Thermal stability of the laccase was determined by incubating the enzyme solution (0,3 g I"1) in 60 mM citrate buffer (pH 6). The residual enzyme activities were measured on ABTS. As shown from the results the half lives of the laccase were 26 and 5.5 hrs at 50, and 60 °C, respectively (Fig. 3).
Specific activity
Specific activities of the purified Tfiielavia laccase were determined towards different laccase substrates. The activities were determined towards ABTS (Niku-Paavola et al, 1988), di-metoxy-phenol (DMP) (Schlosser et al., 1997), syringaldazine (Paszczynski et al., 1985), and guaiacol (Leonowicz & Grzywnowicz, 1981). For ABTS the activity measurements were carried out in 25 mM succinate buffer pH 4.5 at 25°C and for other substrates in 25 mM MES buffer, pH 5.5. Results are shown in Table 1.
Tabla 1. Specific activities of the purified Thlelavia wild type laccase (wt TaLccl) .
Substrata Wt TaLccl alcat/mg
ABTS 1020
DMP 260
syringaldazin 490
Guaiacol 63
Inhibition of the laccase
The effect of various inhibitors on laccase activity was determined by measuring the oxygen consumption during the enzyme reaction with ABTS in sealed and fully filled Erlenmeyer flasks with an Orion Research 081010 oxygen electrode (Software: SensorLink™ PCM800; Orion, Espoo, Finland). The oxygen consumption rates were measured from solutions containing suitable amout of the laccase, 2 xnM ABTS, and various inhibitors in different concentrations, in 25 mM succinate buffer (pH 4.5) in a 30 ml reaction volume.

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Table 2. Inhibition of the Thielavia wild type laccase.
Compound Concentration Inhibition(%) vrt TaLocl
EDTA 10 mM 0
NaN3 0.5 mM 99
KCK 0.1 mM 65
NaCl 0.1 mM 35
NaCl 1 mM 42
N-terminal and internal amino acid sequencing
The N-terminus of the protein as well as the internal peptides were sequenced according to Edman degradation chemistry (Edman and Begg, 1967) using PE Biosystems Procise Sequencer. For peptide preparation, the lyophilized protein was reduced with dithiotreitol, carboxymethylated with iodoacetamide and cleaved with sequencing grade trypsin (Promega) at enzyme/substrate mass ratios 1:100 for 12 hours at 37 C in 0.1 M ammoniumbicarbonate, pH 8.3 (Stone et al., 1988). Generated peptides were separated by reversed-phase high performance liquid chromatography (RP-HPLC, Vydac C-18 column) with a linear acetonitrile gradient (0-60% acetonitrile in 0.1% trifluoroacetic acid). The internal peptide sequences are shown in Table 3. The N-terminus of the protein could not be obtained, because it was presumably blocked.
Table 3. Internal peptide sequences determined from Ttu.ela.vrta. -laccase (ALKO4197) . The N-terminus of the protein was presumably blocked.
Peptide Sequence SEQ ID NO Comments
Pept. 1 Pept. 2 yQG&ENTLPTNOGLPVPNH ENWIGPDGVLK SEQ ID NO: 1 SEQ ID NO: 2 fin equal Ile signal can also be seen in the 12th cycle
Pept. 3 (S)LFLAVGOR SEQ ID NO: 3 (S) result unsure
EXAMPLE 3. Cloning of the Tkielovia arenaria ALKu4i97 gene encoding TaLccl
Standard molecular biology methods were used in the isolation and enzyme treatments of DNA (plasmids, DNA fragments), in E. coli transformations, etc. The basic methods used are described in the standard molecular biology handbooks, e.g. Sambrook et al. (1989) and Sambrook and Russell (2001).

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The genomic library of Thielavia arenaria ALKO4197 was made to Lambda DASH*II vector (Stratagene, USA) according to the instructions from the supplier. The chromosomal DNA, isolated by the method of Raeder and Broda (1985), was partially digested with Sau3A. The digested DNA was size-fractionated in an agarose gel after which the fragments of the chosen size (9-23 kb) were isolated, dephosphorylated and ligated to the BamHI digested lambda vector arms. The ligation mixture was packaged using the Gigapack III XL packaging extracts according to the manufacturer's instructions (Stratagene, USA). The titer of the genomic library was 1.2 x 10s pfu/ml and that of the amplified library was 1.1 x 1010 pfu/ml.
The probe used for screening of the gene bank was amplified by PCR using the Thielavia ALKO4197 genomic DNA as a template. First, several primers (degenerate oligos) were planned (Table 4, SEQ ID NO: 4-9) and tested in PCR reactions. The sequences of the primers based on the ammo acid sequences of the peptides obtained from the purified TaLccl (Fig. 5). The combinations of the primers in the PCR reactions were chosen according to the location of the peptide homologues in the published laccase sequences. The PCR reaction mixtures contained 50 mM Tris-HCl, pH 9.0, 15 mM (NH4)2SO4, 0.1% Triton X-100, 5 % DMSO, 3 mM MgCl2) 0.2 mM dNTPs, 5 uM each primer and 2 units of Dynazyme EXT DNA polymerase (Finnzymes, Finland) and approximately 5 ug of genomic DNA. The conditions for the PCR reactions were the following: 5 min initial denaturation at 95°C, followed by 30 cycles of 1 min at 95°C, 1 min annealing at 50°C, 2 min extension at 72°C and a final extension at 72°C for 10 min. The most specific result and DNA products having the expected sizes (calculated from the published fungal laccase sequences) were obtained from the PCR reactions performed with the primer combinations POX27/POX31 and POX28/POX31. The DNA fragments were isolated from these two reactions and they were cloned to pCR® Blunt-TOPO® vector (Invitrogen, USA). The DNA products were characterized by sequencing and by performing Southern blot hybridizations to the genomic Thielavia DNA digested with several restriction enzymes. The hybridization patterns obtained with the two fragments were identical in stringent washing conditions. The deduced amino acid sequences of both the fragments contained sequences having homology to several published laccase sequences (BLAST program, ^version 2.2.9 af NCBI, National Center for Biotechnology Information; Altschul et aL, 1990).
The 1 kb fragment obtained from the PCR reaction by using the primers POX27 and POX31 was chosen as a probe for screening the gene bank. The sequence of this fragment included the region of the gene that had been amplified by using the primers POX28 and POX31. Also, the deduced amino acid

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sequence included the sequence of the internal TaLccl Peptide 3 (Example 2, Fig. 5). The pCR®Blunt-TOPO* vector containing the PCR fragment was named as pALK1550.
Table 4. The oligonucleotides (SEQ ID NOs: 4-9) synthesized as PCR primers to amplify a proba for screening the Talccl gena from the genomic library. Oligo, oligonudeotide; Oligo location, the aaino acids of the peptide used in planning of the oligonudeotide sequence.
Oligo Length (nts) Degeneracy1 Sequence Peptide"3 Oligo loc.
POX26 26 8 GAGAACTGGATCGGYCCCGAYGGYGT (s) TaLccl 2 1-9
POX27 17 48 GARAAYTGGATHGGXCC (s) TaLccl 2 1-6
POX28 20 16 CTCTTCCTCGCYGTSGGYCA (s) TaLccl 3 2-8
POX29 20 16 TGRCCSACRGCGAGGAAGAG (as) TaLccl 3 2-8
POX30 20 8 TACCAGGGYGCYCCSAACAC (s) TaLccl 1 1-7
POX31 20 8 GTGTTSGGRGCRCCCTGGTA (as) (d TaLccl 1 1-7
(a To reduce degeneracy, some codons were chosen according to fungal preference.
(b D = A or G or T, H - A or C or T, R = A or G, S = C or G, W = A or T, X = I
(inositol) or C, Y = T or C; "s" in the parenthesis = sense strand, wa3" in the
parenthesis = antisense strand.
(c The peptide sequences are included in Fig. 5.
(d The codon usage was chosen according to the preference in the xylanase genes
xynllA, xynllB and xynllC isolated from C. thermophilum ALKO4265 (EMBL AJ508931-
508933) .
The insert from the plasmid pALK1550 was labeled by using digoxigenin according to the supplier's instructions (Roche, Germany). About l.8xl05 plaques from the amplified genomic library were screened. The hybridization temperature for the filters was 68°C and the filters were washed 2x5 min at RT using 2 x SSC - 0.1 % SDS followed by 2 x 15 min at 68°C using 0.1 x SSC-0.1% SDS. Several positive plaques were obtained Some of the positive plaques were giving a strong hybridization but, in addition, there was an amount of plaques hybridizing more weakly to the probe. Five of the strongly hybridizing plaques were purified (Fl - F5) and the phage DNAs were isolated. A Southern blot analysis of the phage DNAs revealed that the clones Fl, F2 and F3 all included an about 3 kb Xhol and about 7 kb BamHL fragments hybridizing to the probe. These fragments, and in addition a 6.1 kb Sacll and 3.8 kb Spel fragments, were isolated from the clone Fl. The fragments were ligated to pBluescript II KS+ or SK+ vector (Stratagene, USA) and the plasmids obtained were named as pALK1606 (Xhol fragment), pALK1607 (BamHl fragment), pALK1341 (SacU fragment) and pALK1342 (Spel fragment). The gene encoding the TaLccl was sequenced from these clones and from the shorter subclones isolated from pALK1607. The full-length Talccl gene was included in the plasmids pALK1607, pALK1341 and pALK1342. The E.coli strain RF5473 including the plasmid pALK1342 was deposited to the DSM collection (DSM 15484).

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The Talccl sequence (SEQ ID NO: 11) and the deduced amino acid sequence (SEQ ED NO: 12) are shown in Fig. 6. The length of the gene is 2279 bp (including the stop codon). Six introns were found having lengths of 51, 62, 91, 83, 79 and 59 bps. The deduced protein sequence consists of 617 amino acids including a predicted signal sequence of 21 amino acids (SignalP V2.0; Nielsen et a!., 1997 and Nielsen and Krogh, 1998) and a "tail" sequence of 13 amino acids (starting after the sequence DSGL). The peptides purified from the wt TaLccl were all found from the deduced amino acid sequence indicating that the gene cloned encodes the laccase purified from ALKO4197. The predicted molecular mass was 64456 Da for the mature polypeptide and the predicted pi was 6.31 (amino acids 22 - 604, signal sequence and the C-terminal tail removed). These predictions were made using the Compute pI/MW tool at ExPASy server (Gasteiger et al., 2003). The deduced amino acid sequence included nine putative N-glycosylation sites. The homologies to the published laccase sequences were searched using the BLAST program, version 2.2.9 at NCBI (National Center for Biotechnology Information) (Altschul et al., 1990). The highest homologies were found to the laccases from Melanocarpus albomyces. Podospora anserina and Neurospora crassa (EMBL accession numbers CAE00180.1, LAC2PODAN, XP_323881.1). The TaLccl sequence was aligned with these three laccase sequences, with the other sequences from the NCBI database having over 50% identity to TaLccl in the BLAST search, and with the laccase sequences from Myceliophthora thermophila and Scytalidium thermophilum, found from the patent database (EP0765394 Bl/US 5,981,243 and US 5,750,388, respectively). The identity values obtained by using Needleman-Wunsch global alignment (EMBLOSUM62, Gap penalty 10.0, Extend penalty 0.5; EMBOSS program package, version 2.9.0) are shown in Table 5.
Table 5. The identity values (%) obtained £roa Needleman-Wunsch global alignment: of the deduced laccase amino add sequences. The full-length amino acid sequences including the signal peptides were aligned. Matrix: EMBLOSUM62, Gap penalty 10.0, Extend penalty 0.5. Mali Melanocarpus albomyces CAE001810, Mth Myceliophthora thermophila laccase from EP 0765394 Bl, Pan Podospora anserina LAC2_PODAN, 5th Scytalidium thermophilum laccase from OS 5,750,388, Ncr LAC1 Neurospora crassa LAC1 NEUCR. Ncr XP N. crassa XP 323BB1, Ncr iCSNCLO- tf. cras-sa KSNC£&, Ncr LAC2 N. crassa LAC2_NEUCR, Cpa Cryphonectria parasitlca LAC1_CRYPA, Ggr Gaeumannomyces graminis var tritici Lac3 CAD100749.

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EXAMPLE 4. Production of the recombinant TaLccl io Trichoderma reesei
The expression plasmid pALK1667 was constructed for production of recombinant TaLccl in Trichoderma reesei. The Talccl gene with its own signal sequence was exactly fused to the T. reesei cbhl (cel7A) promoter by PCR. The cbhl promoter, cbhl terminator, amdS marker and the cbhl 3' flanking region included were as described in Paloheimo et al. (2003). The Talccl gene fragment was excised from its 3'-end by NcdL This cleavage left 80 bp of the Talccl terminator in the construct, prior to the cbhl terminator sequence. The 10.1 kb linear expression cassette (Fig. 7) was isolated from the vector backbone after EcoRI digestion and was transformed to T. reesei A47 protoplasts. The transformations were performed as in Penttila et al. (1987) with the modifications described in Karhunen et al. (1993). The transformants were purified on selection plates through single conidia prior to sporulating them on PD.
The laccase production of the transformants was analysed from the culture supematants of the shake flask cultivations (50 ml). The transformants were grown for 7 days in a complex lactose-based cellulase-indueing-medium-(Joutsjoici"et alrl993)^uSerecfwith 5% KH2PO4 and supplemented with 0.1 mM CuSO4 at pH 6.0. The laccase activity was assayed using ABTS as a substrate as described in Example 1. The possible targeting of the expression cassette to the cbhl locus was screened as a CBHI-negative phenotype by dot blot (Minifold I-SRC 96; Schleicher & Schuell, Dassel, Germany) or by Western blot The detection of the CBHI protein was performed using the monoclonal antibodies CI-258 or CI-261 (Aho et al., 1991) and the ProtoBlot Western blot AP system (Promega). The genotypes of the

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chosen transformants were confirmed by using Southern blots in which several genomic digests were included and the respective expression cassette was used as a probe.
The chosen CBHI-negative transformants RF5597 and RF5598 were fermented to obtain material for purification of the recombinant TaLccl (Example 5) and for the application tests (Examples 7-12).
EXAMPLE 5. Purification of TaLccl of Thielavia (ALKO4197) expressed in T. reesei
TaLccl is the multiple pi isoform laccase i.e. the enzyme shows 6-7 distinct bands in isoelectric focusing and subsequent active staining within the pH range of 5.5-6.9. The buffer of culture supernatant was first changed to 5 mM Tris-HCl buffer, pH 8.5 with Sephadex G25 resin. The sample was loaded onto a pre equilibrated DEAE Sephadex FF column. Proteins were eluted with a linear Na2SO4 salt gradient (0-350 mM). Laccase positive fractions from DEAE Sepharose were pooled and the buffer changed to 5 mM Tris-HCl buffer, pH 8.5 with Pharmacia PD 10 columns. The sample was loaded on a pre equilibrated Resource Q column. Proteins were eluted with an increasing linear Na2SO4 (0-200 mM). Laccase positive fractions eluted within 5-40 mM Na2SO4 salt concentration. The final purification step was carried out with gel filtration. The laccase positive fractions from Resource Q were pooled and concentrated with ContraSep MWCO 10 kDa. The sample was loaded on a pre equilibrated Sephacryi S-100 gel filtration column. The buffer used in the gel filtration was 100 mM Tris-HCl, pH 7.3, containing 150 mM NaCl. The SDS-PAGE showing the purification of the recombinant TaLccl laccase is shown in Fig. 4.
EXAMPLE 6. Characterization of the recombinant TaLccl laccase
The purified recombinant Thielavia arenaria TaLccl was characterized in terms of pH optimum, thermal stability, and pi as described in Example 2. The molecular weight was determined by MALDI-TOF mass spectrometry on a Ultrafiex™ time-of-fiight instrument (BrukerDaltonics, Germany) as previously described {Paiooea et a/.72O03). The redox potential oTTaLccl was also determined with a combined Pt-AgCl/KCl microelectrode at pH 5.0 according to Sigoillot et al (2004)
The characterization results are collected to Table 6.

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Table S. Summary of the characteristics of the recombinant Ttd.ela.Yla arenarxa TaLccl (rTaLccl) and the wild type TaLccl (wt TaLcc) Iacca3ea. nd = not determined.
Laccase pH optimum on guaiacol T4(60°C) (hrs) Pi Number ofPi isofoxma MW(MALDX-TOF) E° mV
rTaLccl 6.0 5 5.5-6.9 6-7 71 890 560
wt TaLcc 6.0 5.5 5.5-6.9 6-7 Nd nd
The inhibition effect of different compounds on the activity of the recombinant TaLccl laccase was determined as described in Example 2. Results are shown in Table 7.
Table 7. Inhibition of the recombinant Thielavia aranaria TaLccl laccase (rTaLccl) activity by various compounds. Inhibition was tested by oxygen consumption measurements in standard condition using ABTS as substrate (Example 2). As a comparison the inhibition results of the wild type Thielavia arenarxa laccase (wtTaLcc) are also shown, nd » not determined.
Compound Concentr. Inhibition (%)of laccase
(nM) wtTaLca rTaLccl
EDTA 10 0 5
NaN3 0.5 99 95
KCN 0.1 65 60
KCti 1 Nd 90
NaCl 0.1 35 0
NaCl 1 42 0
Specific activities of the purified TaLccl were determined towards ABTS, dimetoxy phenol (DMP), syringaldazine, and guaiacol as described in Example 2. The ABTS activity measurements were carried out in 25 mM succinate buffer pH 4.5 at 25°C, and the other activities in 25 mM M£S buffer, pH 5.5. The results are shown in Table 8.
Table 3. Specific activities of recombinant TaLccl (rTaLccl) compared to the specific activities of the wild type enzyme (wtTaLcc).
Substrate Spec. act. wtTaLcc (nkat/mg) Spec. act. rTaLccl (nkat/mg)
ABTS 1020 910
DMP 260 285
Syringaldazine 490 340
Guaiacol 63 61

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The biochemical data presented here clearly indicates that the recorabinant TaLccl is the same protein as the wild type Thielavia laccase purified from the culture supernatant
Kinetic parameters of Thielavia and Melanocarpus laccases
The kinetic parameters, Michaelis-Menthen constant Km, turn-over number kcat and the specificity constant (kcat/Km) were determined on ABTS and 2,6-dimethoxy phenol (DMP), and syringaldatzin. The measurements on ABTS were done in 25 mM succinate buffer, pH 4.5. On syringaldazin and DMP 40 mM MES buffer, pH 6 and 25 mM succinate buffer, . All activity assays were carried out at 25°C. Kinetic parameters were estimated by a nonlinear regression curve fit The results are shown in Table 9. The values were compared to those of Melanocarpus albomyces MaL, laccase.
Table 9. Kinafcic parameters of recombinant TaLccl determined on ABTS, syringaldazixx, and DMP. A3 a comparison the values of a well-known laccase from Melanocarpus albomyces MaL are also shown.
TaLccl MaL
ABTS
K»(HM) 75 270
kcat (min-1) 4130 4690
kct/Km(M-1min-1) 5.51*10' 1.8*107
me
K»(HM) 17 5
kMt (min'1) 4030 4160
lccat/K,.(M"iinin'1) 2.37*10" 8.1*10"
4.3 1.3
1940 4710
kcat/km(M-1 min-1) 4.51*108 3.6*109
EXAMPLE 7. Performance of the TaLccl laccase preparation in denim bleaching at different pH-valaes
Recombinant TaLccl laccase preparations produced using Thrichoderma as a host were used in all the application tests, in Examples 7 - 12. The TaLccl preparation (derived from the strain RF5598) was tested for its ability to bleach denim and compared to a commercial laccase preparation DeniLite II Base from Novozymes.

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Lee Cooper jeans (Hamilton, 111-1060-55522-65, MASI Company Oy, Finland, former name M.A.S.I. jeans Oy) that were made of ring spun yarn in warp and open-end yam b weft and were previously desized and treated with neutral ECOSTONEw cellulase were used as a test material. Laccase treatments were performed in LP-2 Launder Ometer as follows. About 10 g of denim swatches (15 x i4 cm) were loaded into 1.2 liter containers containing 200 ml Me Evaine's citrate phosphate buffer pH 5, 6 or 7 and the containers were temperated. Enzyme with or without the mediator (methyl syringate, DeniLite II Assist, Novozymes) was added. Enzyme was dosed 200 nkat/g and the mediator 10 mg/g on the weight of fabric. The enzyme activity was measured with ABTS substrate as in Example 1 but using citrate phosphate buffer in all examples 7-12. The Launder Ometer was run at 50°C for 30 min and after that the temperature in Launder was raised to 80 °C for 10 min. The swatches were rinsed carefully with warm water, dried half-dry in a tumbler and after that air dried.
The bleaching effect was evaluated by measuring the colour as reflectance values with the Minolta Spectrophotometer CM 1000 (Minolta Co.) using L*a*b* color space coordinates (illuminant D65/2°). The colour from both sides of the swatches was measured before and after the laccase treatment Each measurement was the average of several, at least ten, measurements.
Table 10 and Fig. 8 clearly show that TaLccl laccase was superior in decolorization of indigo dye of denim compared to the commercial DeniLite H Base at all pH values from 5 to 7, pH 6 being the optimum. Only the TaLccl preparation was capable in achieving a strongly bleached look with the highest lightness value. The increase of lightness and the decrease of blueness on the reverse side of denim was also the highest with denim treated with TaLccl laccase preparation and the mediator. Without the mediator the laccases did not have a notable effect on denim. (Table 11). Bleaching tests performed with TaLccl laccase later showed that the enzyme works well at broad pH range (4-8).

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Table 10. Colour measurements of the face side of Honim treated with laecaso preparations and the mediator in Launder at pH 5 — 7. L* indicates lightness, -b* is the blue direction, +b* is the yellow direction.
Prap. Enzyme Mediator Before laccase Treatment After laccase Treatment Increase
rkat/g mg/g Conditions L* b* L* B* of L*
TaLccl 200 10 30 min, 50*C, pH 5 28.52 -18.46 42.04 -14.7B 13.52
DeniLite 200 10 30 min, 50°C, pH S 28.41 -18.70 35.70 -17.59 7.29
TaLccl DeniLita 200 200 10 10 30 min, 50°C, pH 6 30 min, 50°C, pH 6 27.7B 26.98 -18.49 -18.67 48.97 34.16 -12.36-17.82 21.19 7.18
TaLccl 200 10 30 min, 50°C, pH 7 27.79 -19.00 44.71 -14.43 16.92
DeniLite 200 10 30 min, 50°C, pH 7 28.67 -18.99 34.51 -17.75 5.84
Table 11. Colour measurements of tha face sida of denim treated with laccase preparations without the mediator or mediator only in Launder at pH 5 — 7. L*
indicates lightness, -b* is the blue direction, +b* is the yellow direction.
Prep. Enzyme nfcat/g Mediator mg/g Conditions Before laccasa Treatment: L* b* After laccase Treatment 1* B* Increase of I.*
TaLccl 200 0 30 min, 50"C, pH 5 28.08 -18.41 28.22 -18.35 0.14
DeniLite 200 0 30 min, 50°C, pH 5 29. IB -18.55 29.30 -18.34 0.12
Mediator TaLccl 0 200 10 0 30 min, 50°C, pH 5 30 min, 50"C, pH 6 29.85 28.77 -18.58 -18.77 29.90 29.58 -18.15 -18.38 0.05 0.82
DeniLite 200 0 30 min, 50*C, pH 6 28.55 -18.48 28.77 -18.52 0.22
Mediator 0 10 30 min, 50*C, pH 6 28.68 -18.40 28.90 -18.37 0.22
TaLccl 200 0 30 min, 50"C, pH 7 27.43 -18.93 27.B0 -18.50 0.37
DeniLite 200 0 30 min, 50"C, pH 7 27.82 -18.93 29.78 -18.31 1.96
Mediator 0 10 30 min, 50°C, pH 7 29.00 -18.94 30.05 -18.46 1.0S
EXAMPLE 8. Performance of TaLccl laccase preparation in denim bleaching at different temperatures—

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The test system and denim were as in Example 7, except that the conditions during the laccase and mediator treatment in Launder were 30 min, pH 6 and temperature 30 - 80°C. Also, the enzyme was inactivated by an alkaline treatment instead of raising the temperature in Launder as follows. After removing swatches from the containers they were soaked in warm water containing NaOH (pH 11.5) for 10 min and rinsed carefully with warm water. The swatches were dried half-dry in a tumbler and after that air dried. The bleaching effect was evaluated by measuring the colour as reflectance values as in Example 7.
Table 12 and Fig. 9 show that TaLccl laccase was superior in bleaching of denim (higher increase of lightness) compared to DeniLite II Base at 40 - 80 °C. The temperature 60 - 70 °C was the most optimal for TaLccl and the look of the denim fabric was strongly faded. The DeniLite II Base was capable only for moderate decolorization of indigo.
table 12. Colour measurements of the face si-da of denim treated with laccases and the mediator in Launder at different temperatures. L* indicates lightness, -b* is the blue direction, +b* is the yellow direction.
Beep. Enzyme Mediator Before lacoasa Treataant After laccase Treatment Increase
nkat/g ssg/g Conditions L* B* of !¦*
TaLccl 200 10 30 min, 30*C, pH 6 29.58 -18.82 33.68 -19.17 4.10
DeniLite 200 10 30 min, 30°C, pH 6 29.56 -18.60 32.73 -18.77 3.17
TaLccl DeniLite 200 200 10 30 min, 40"C, pH 6 10 30 min, 40°C, pH 6 28.81 28.87 -19.03 -18.90 37.49 32.94 -18.64 -19.14 8.68 4.07
TaLccl 200 10 30 min, 50"C, pfl 6 28.40 -18.88 42.99 -17.06 14.59
DeniLite 200 10 30 min, 50"C, pH 6 28.41 -19.10 34.67 -19.07 S.26
TaLccl 200 10 30 min, 60"C, pH 6 29.14 -19.03 47.56 -14.55 18.42
DeniLite 200 10 30 min, 60'C, pH 6 29.06 -18.99 35.92 -18.33 6.86
TaLccl 200 10 30 min, 70°C, pH 6 29.09 -19.03 46.94 -13.82 17.85
DeniLite 200 10 30 min, 70"C, pH 6 29.05 -19.15 36.72 -17.35 7.67
TaLccl 200 10 30 min, 80*C, pH 6 29.39 -19.01 39.24 -15.74 9.85
TaLccl' 200 0 30 min, 80"C, pH 6 29.63 -19.16 30.27 -IB.62 0.64
DeniLite 200 10 30 min, 80°Cf pH 6 29.28 -18.97 35.33 -17.02 £.05
DeniLite 200 0 30 min, 80"C, pH 6 29.81 -18.74 31.69 -18.19 1.88 .
EXAMPLE 9. Effect of enzyme dosage in bleaching of denim with laccase-mediator system
Lee Cooper Jeans previously washed with ECOSTONE® cellulases (Example 7) were treated with a TaLccl laccase product from Trichoderma strain RF5598 and DeniLite Base from Novozymes using different laccase dosages. The laccase treatments were performed with Electrolux's Wascator FOM 71

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CLS washer extractor under optimal conditions for each enzyme, shown in Table 13. Methyl syringate (Example 7) was used as a mediator. Laccase was inactivated after draining by raising the pH above 11 by NaOH (10 min, 60°C) and rinsing 3 times. The fabrics were dried in a tumbler.
Table 13. The process parameters used in tha bleaching -test with TaLccl and DeniLita II Base laccase preparations with different dosages.
Process parameter TaLccl laccase DeniLite II Base laccase
Denim load 1.5 kg 1.5 kg
Water 15 1 15 1
Buffer/pH control 37.5 g Na2HPO,*2H20 14.7 g citric acid Acetic acid
pH 6 5 -5,5
Time 30 min or 60 min 30 min
Temperature 60"C 60°C
Enzyme dosage 20 or 100 nkat/g fabric 20 or 100 nkat/g fabric
Mediator Dosage 1 or 5 mg/g fabric 1 or 5 mg/g fabric
The bleaching effect was evaluated by measuring the colour after laccase treatment as reflectance values (as in Example 7) and comparing the values to previously measured, values after celMase treatment Results in Table 14 and Fig. 10 show that increase of the dosage greatly improved the bleaching performance of TaLccl laccase.
With DeniLite II Base the effect of the dosage was low. Increasing the time from 30 min to 60 min further improved the performance of TaLccl laccase. With DeniLite increasing the treatment time longer than 30 minutes provides no additional bleaching performance as discussed in Mueller and Shi (2000). With TaLccl laccase it was possible to achieve very strong bleaching effect that is normally obtained only with the use of high amounts of sodium hypochlorite. Also the abraded look was maintained. Up to this date such high increase of lightness of denim obtained with the use of a laccase-mediator system has not been reported.

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Table 14. Colour measurements of the faca side, of deni.fi treated with laccaaa preparations and the mediator in Lannder using different dosages. L* indicates lightness, -b* is the blue direction, +b* is the yellow direction.
Prep. Enryme rkat/g Mediator mg/g Conditions Before laccasa Treatment L* b* After laccase treatmentL* B* Increaseof L*
TaLccl 20 1 30 min, 60*C, pH 6 28.37 -13.56 40.61 -16.16 12.24
TaLccl TaLccl TaLccl 100 20 100 5 1 5 30 min, 60°C, pH S 60 min, 60"C, pH 6 60 min, 60'C, pH 6 27.96 27.63 27.73 -18.BB -18.54 -18.91 49.64 42.72 53.46 -13.75 -15.61 -11.64 21.38 15.09 25.68
DeniLite 20 1 30 min, 60*C, pH 5.3 28.78 -18.46 36.13 -17.12 7.35
DeniLite 100 5 30 min, 60'C, DH 5-5.5 28.88 -19.74 37.19 -17.37 8.32
EXAMPLE 10. Bleaching of denim with laccase-mediator system compared to bleaching with sodium hypochlorite
Different types of demin were treated with a TaLccl laccase product from Trichoderma strain RF5597 and DeniLite II Base from Novozymes and the results obtained were compared to hypochlorite bleaching. All together 27 pieces of different kinds of denim (whole jeans), which had been previously washed with cellulase after desizing to different abrasion levels or had been only desized with ECOSTONE®A200 (60°C, 10 min), were collected for the test (Table 15). The jeans were made of Indigo dyed denim, except the English jeans contained Indigo dyed denim with sulphur bottom. Jeans were cut in such a way that pieces from the same pair of jeans could be used for all tests.
The jeans were made of Indigo dyed denim (right hand twill), except the English jeans contained Indigo dyed denim with sulphur bottom. The weights of the original (i.e. not desized) denim fabrics were typically higher than 475 g/m2 (14 oz/yd2). The weights of three 10 cm x 10 cm pieces were measured after 24 hours incubation at room temperature (ca. 22 C and at relative moisture ca. 64%).

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Table 15, Denim samples used in the bleaching test.
Sample Denim type Source of denim SniyTtto used in the cellulase treatment
Tl Tincan jeans MASI Company Oy, Finland Desized only, not cellulase treated
T2 Tiacan jeans MASI Company Oy, Finland Ecostone N400, AB Enzymes
T3 Tincan jeans MA5I Company Oy, Finland Ecostone L300, AB Enzymes
T4 Tincan jeans MASI Company Oy, Finland Denimax 991S, Novozymes

LI Lee Cooper jeans MASI Company Oy, Finland Desized only, not cellulase treated
L2 Lee Cooper jeans MASI Company Oy, Finland Ecostone L, AB Enzymes
L3 Lee Cooper jeans MASI Company Oy, Finland Biotouch C29, AB Enzymes
L4 Lea Cooper jeans MASI Company oy, Finland Ecostone L, AB Enzymes
L5 Lee Cooper jeans MASI Company Oy, Finland Indiage Max L, Genencor International
L6 Lee Cooper jeans MASI Company Oy, Finland Indiage Super L, Genencor International
L7 Lea Cooper jeans MASI Company Oy, Finland Ecostona CXP500 Exper., AB Enzymes
L8 Lee Cooper jeans MASI Company Oy, Finland Ecostone CXP experimental, AB Enzyme3
L9 Lee Cooper jeans MASI Company Oy, Finland Ecostone N400, AB Enzymes

Wl Warric jeans Iki-Asu Oy, Finland Desized only, not cellulase treated
W2 Warric jeans Iki-Asu Oy, Finland Ecostone P4500, AB Enzymes
W3 warric jeans Iki-Asu Oy, Finland Indiage Max G, Genencor International
W4 Warric jeans Iki-Asu Oy, Finland Ecostone NP2000, AB Enzymes
W5 Warric jeans Iki-Asu Oy, Finland Ecostone 1.900, AB Enzymes
W6 Warric jean3 Iki-Asu Oy, Finland Denabraide MBL 65 E, logen

El English jeans English company Desized only, not cellulase treated
E2 English, jeans English company Ecostone NP200O, AB Enzymes
E3 English jeans English company Rocksoft NCE-EA, Dyadic
E4 English jeans English company Rocksoft NCE-2X, Dyadic
E5 English jeans English company Ecostone L900, AB Enzymes
E6 English jeans English company Ecostone L900, AB Enzymes
E7 English jeans English company Ecostone L-9QO, AB Enzymes

Bl Basic Jeans MASI Company Oy, Finland Ecostone P1250, AB Enzymes
The laccase treatments were performed with Electrolux's Wascator FOM 71 CLS washer extractor under optimal conditions for each enzyme, described in Table 16. Methyl syringate (Example 7) was used as a mediator. Laccase was inactivated after draining by raising the pK above 11 by NaOH (10 rain, 50°C) and rinsing for three times. The fabrics were dried in a tumbler.

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Table 16. The process parameters used in the bleaching test with TaLccl and DeniLite II Base laccase preparations.

The sodium hypochlorite bleaching was performed with Electrolux's Wascator FOM 71 CLS washer extractor vising denim obtained from the same jeans as in laccase treatments under conditions described in Table 17. Sodium hydroxide was added before adding 25 ml/I of 10 % sodium hypochlorite solution (Klorite Forte, Farmos Oy) to keep the pH above 10.5 during the treatment. After draining the bleach liquor, there was one 2 minutes rinsing step with the liquor ratio of 1:20 before dechlorinating with sodium thiosulphate. After dechlorinating the denim samples were rinsed 3 times for 2 minutes with the liquor ratio of 1:20. The fabrics were dried in a tumbler.
Table 17. The process parameters used in the sodium hypochlorite bleaching and dechlorinating.

The bleaching effect was evaluated by measuring the colour as reflectance values as in Example 7. The results obtained are shown in Tables 18-20 and Fig. 11. The TaLccI laccase was superior compared to

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DeniLite II Base laccase (ca. 55 - 200 % better) and sodium hypochlorite bleaching (even 60 - 115 % better) under above mentioned conditions with all cellulase treated denim samples of each type. With fabrics that had only been desized the bleaching effect (increase of L* on the face side of denim) obtained with TaLccl preparation was equal or better than with the sodium hypochlorite and over 100 % better than with DeniLite II Base. Different looks were obtained depending on the type of the denim and cellulase treatment used. "Sulphur-bottom type" denim was the most difficult to bleach.
Tsbla 18, Colour measurements of the face side of ctonia treated with TaLcd laccase preparation (RF5597) . L* indicates lightness, -b* is the blue direction, +b* is the yellow direction.
Sample No. Before laccase Treatment After laccase treatment Increase
L* b* 1* b* of L*
Tl 25.02 -12.75 34.82 -16.36 9.80
T2 28.20 -16.20 42.70 -14.60 14.50
T3 29.08 -17.04 47.09 -14.04 18.01
T4 27.16 -16.26 43.53 -15.18 16.37

Ll 20.21 -16.14 33.13 -19.41 12.92
L2 31.81 -19.08 50.89 -13.17 19.08
L3 28.45 -19.38 47.66 -14.82 19.21
L4 29.42 -19.31 48.39 -13.83 18.97
L5 28.49 -18.82 47.73 -14.28 19.24
L6 29.54 -18.64 46.71 -14.68 17.17
L7 27.20 -19.02 42.95 -16.23 15.75
L8 29.46 -19.04 46.73 -14.86 17.27
L9 28.80 -18.73 45.02 -14.91 16.22

Wl 18.03 -13.04 28.54 -17.55 10.51
W2 26.12 -17.08 42.16 -14.48 16.04
W3 28.55 -16.63 44.08 -13.66 15.53
W4 26.22 -16.13 39.54 -14.72 13.32
W5 23.97 -17.54 40.49 -15.38 16.52
we 24.01 -17.01 39.58 -15.62 15.57

El 17 . "47 " " " -10". 05 22.71 -13.97" 5.24
E2 25.48 -14.75 35.99 -14.08 10.51
E3 25.26 -15.91 36.52 -14.46 11.26
E4 27.04 -15.91 38.79 -13.94 11.75
E5 25.19 -15.32 37.53 -14.03 12.34
E6 22.54 -14.90 34.17 -14.79 11.63
E7 22.84 -15.72 36.00 -14.95 13.16

Bl 29.48 -14.67 47.19 -12.75 17.71

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Table 19. Colour measurements of the face sida of denim treated with DeniLite XX Base lacc»36. L* indicates lightness, -b* is the blue direction, +b* is the yellow
direction.
Sample No. Before laccase Treatment After laccase Treatment Increase
L* b* L+ b* of L*
Tl 24.66 -12.76 28.84 -15.02 4.18
T2 28.54 -15.97 33.34 -16.19 4.80
T3 29.78 -16.94 37.86 -16.80 8.08
T4 27.68 -15.70 35.00 -16.79 7.32

11 19.99 -16.35 25.75 -IB.94 5.76
12 32.70 -18.39 43.80 -15.74 11.10
13 28.67 -18.94 39.27 -17.47 10.60
14 29.40 -19.40 40.28 -17.19 10.88
15 28.29 -19.10 39.14 -17.26 10.85
16 29.74 -18.84 40.48 -16.45 10.74
n 27.70 -18.73 35.54 -18.26 7.84
L8 29. B6 -18.71 40.39 -16.80 10.53
I>9 28.95 -18.71 38.85 -16.89 9.90

Wl 18.67 -12.81 23.04 -16.09 4.37
B2 26.62 -16.80 35.70 -15.85 9.08
W3 28.06 -16.83 36.90 -15.48 8.84
W4 26.42 -16.27 33.25 -15.53 6.83
W5 24.28 -17.43 33.47 -16.62 9.19
W6 24.32 -17.14 33.00 -16.66 8.68

El 16.94 -9.80 19.11 -12.36 2.17
E2 25.49 -14.17 31.54 -14.34 6.05
E3 24.97 -15.82 32.23 -14.90 7.26
E4 27.47 -15.23 34.26 -14.76 S.73
E5 24.42 -15.68 32.37 -15.02 7.95
E6 22.58 -14.91 29.30 -15.11 6.72
E7 22.76 -15.56 30.73 -15.26 7.97

Bl 28.65 -14.84 36.29 -14.95 7.64

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Table 20. Colour measurements of the face side of denim treated with sodium hypochlorite. L* indicates lightness, -b* is the blue direction, +b* is the yellow direction.
Sample Ho. Before laccase Treatment After laccase Treatment Increase
L* b* L* b* of L*
Tl 25.02 -12.87 33.97 -17.95 8.95
T2 27.65 -16.05 34.38 -18.22 6.73
T3 29.09 -16.80 40.33 -18.30 11.24
T4 27.56 -16.00 37.66 -18.58 10.10

LI 20.54 -16.12 31.23 -20.83 10.69
L2 31.14 -19.03 44.11 -18.60 12.97
L3 28.45 -18.64 39.71 -19.67 11.26
L4 29.15 -19.26 40.63 -19.25 11.48
L5 28.00 -19.01 39.47 -19.48 11.47
L6 29.08 -18.61 41.50 -18.68 12.42
L7 26.76 -19.03 37.91 -19.85 11.15
L8 28.76 -18.67 40.74 -19.12 11.98
L9 28.91 -18.40 40.82 -18.82 11.91

Wl 17.58 -13.07 27.75 -18.76 10.17
W2 25.59 -17.16 39.35 -17.39 13.76
W3 28.12 -16.57 41.11 -16.79 12. S3
W4 25.78 -16.11 38.07 -17.33 12.29
W5 23.72 -17.39 37.12 -18.03 13.40
W6 23.75 -16.81 36.65 -17.95 12.90

El 16.76 -10.10 21.91 -15.03 5.15
E2 25.49 -14.17 33.30 -16.43 7.36
E3 24.73 -15.52 34.13 -16.73 9.40
E4 26.55 -15.56 35.33 —16.86 9.28
E5 24.57 -15.48 33.85 -16.48 9.28
E6 22.20 -14.48 30.61 -16.65 8.41
S7 22.74 -15.52 32.36 -16.75 9.62

Bl 28.35 -15.21 40.62 -17.37 12.27
EXAMPLE ii. Stain removal with iaccases
The TaLccl (RF5598) and Denilite II Base laccase preparations were tested for their ability to remove stains. The following artificially soiled test cloths were used: grass soiling (Art 154, EMPA Testmaterialen, Germany), tea soiling (Art 167, EMPA Testmaterialen, Germany). The fabric was cut in 5.8 x 5.8 cm swatches. Laccase treatments were performed in LP-2 Launder Ometer as follows.

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About 5 g of soiled fabrics were loaded into 1.2 liter containers containing 150 ml Me IIvaine's citrate phosphate buffer pH 6 and the containers were temperated. Enzymes with or without the mediator (methyl syringate, DeniLite II Assist, Novozymes) were dosed as laccase activity units (Example 7). Enzyme dosage was 200 nkat/g and mediator dosage 10 mg/g of the weight of fabric, except at 40°C also dosages of 20 nkat/g and 2 mg/g were used. The Launder Ometer was run at 40, 50 or 60°C and pH 6 for 60 min. After that the swatches were carefully rinsed under running water and in shake flasks containing warm water and dried in the air.
The stain removal effect was evaluated by measuring the colour as reflectance values using L*a*b* color space coordinates (Example 7). The colour of the swatches was measured before and after the laccase treatment
The results of the stain removal tests are shown in Table 21, Table 22 and Figs. 12 -15. The TaLccl preparation was more effective in removal of grass soiling with the mediator at 60°C (highest a*-value = least green, highest lightness L*) than DeniLite II (Fig 12, Table 21). The result looked the best also by visual estimation. TaLccl was slightly better than DenLite at 40°C too (Fig. 14, Table 22). Without the mediator the efficiency in removal of grass stain was low.
TaLccl laccase was also more efficient in removal of tea stain at 60°C with the mediator than DeniLite, mat can be seen in the highest lightness and the lowest redness values in Fig 13 and Table 21 and also by visual estimation. TaLccl was slightly better at 40°C, especially with higher dosage, too (Fig. 15, Table 22). Without the mediator the laccases did not have a notable effect on tea stain. Removal of grass and tea stains was more difficult at 40°C than at higher temperatures.

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EXAMPLE 12. Decolorization of dyes using TaLccl laccase preparation
The TaLccl laccase preparation from strain RF5597 was tested for its ability to decolourize different dyes in the presence or absence of the methyl syringate mediator (Example 7). The experiments were carried out in 250 ml shake flasks containing 100 nil of dye dissolved in citrate phosphate buffer pH 6. Dye concentration 12 mg/100 ml was used. Enzyme was dosed 200 nkat (or 20 nkat) per 100 ml and the mediator 10 (or 1) mg per 100 ml. The shake flasks were incubated at 50°C for 30, 90 and 150 minutes. Samples of 3.5 ml were taken in test tubes for visual evaluation.
The results in Table 23 show that TaLccl laccase is able to decolorize Indigocarmine, Remazol Brilliant Blue (Reactive Blue 19) and Cibacron Brilliant Red 3B-P to great extend and Pontamine Bast Orange to some extend in the presence of the mediator. Decolourization of Indigocarmine was very fast, and the blue colour had turned to light yellow earlier than in 30 min. The reaction seemed to be completed in 90 min with all dyes, since no changes in the colours of the samples was detected after a prolonged incubation.
Table 23. Decolorization of dyes with TaLccl (RF5597) laccasa preparation.
Treatment time 30 and 60 min. - no visually detectable change, + visually-detectable fading of the colour, ++ considerable fading of the colour, +++ complete/almost complete decolorization.


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REFERENCES
Aho S, V Olkkonen, T Jalava, M Paloheimo, R Buhler, M-L Niku-Paavola, EH Bamford and M Korhola. 1991. Monoclonal antibodies against core and cellulose-binding domains of Trichoderma reesei cellobiohydrolases I and II and endoglucanase L Eur. J. Biochem. 200:643-649.
Altschul SF, W Gish, W Miller, EW Myers and DJ Lipman. 1990. Basic local alignment search tool. J.MoLBiol. 215:403-410.
Edman P and G Begg. 1967. A protein sequenator. Eur. J. Biochem. 1:80-91.
Gasteiger, E, A Gattiker, C Hoogland, I Ivanyi, RD Appel and A Bairoch. 2003. ExPASy: the proteiomics server for in-depth protein knowledge and analysis. Nucleic Acids Res. 31:3784-3788.
Joutsjoki, W, TK Torkkeli, and KMH Nevalainen. 1993. Transformation of Trichoderma reesei with the Hormoconis resinae glucoamylase P (gamP) gene: production of a heterologous glucoamylase by Trichoderma reesei. Curr. Genet 24:223-228.
Karhunen T, A Mantyla, KMH Nevalainen, and PL Suominen. 1993. High frequency one-step gene replacement in Trichoderma reesei. L Endoglucanase I overproduction. MoL Gen. Genet. 241:515-522.
Kiiskinen, L-L, M Ratto and K Kruus. 2004. Screening for novel laccase-producing microbes. J. Appl. Microbiol. 97:640-646.
Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680 - 685.
Leonowicz, A and K Grzywnowicz. 1981. Quantitative estimation of laccase forms in some white-rot-fungi using syringaldazine as a substrate. Enzyme Microb. TechnoL 3:55-58.
Lowry OH, NJ Roseborough, AL Fair and RJ RandaiL 1951. Protein measurement with the Folin phenol reagent J. Biol Chem 193: 265-275.
Malardier L, MJ Daboussi, J Mien, F Roussel, C Scazzocchio and Y Brygoo. 1989. Gene 15:147-156.
Mueller M and C Shi. 2000. Laccase for decorization of indigo in denim processing, AATCC, 2000 International conference and exhibition.
Nielsen H, J Engelbrecht, S Brunak and G von Heijne. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engineering 10:1-6.

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Nielsen H and A Krogh. 1998. Prediction of signal peptides and signal anchors by a hidden Markov model. In: Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology (ISMB 6), AAAI Press, Menlo Park, California, pp. 122-130.
Niku-Paavola M-L, E Karhunen, P Salola, V Raunio. 1988. Ligninolytic enzymes of the white-rot fungus Phlebia radiata. Biochem J 254: 877-884.
Paioheimo M, A Mantyia, J Kaiiio, and P Suominen. 2003. High-yield production of a bacterial xylanase in the filamentous fungus Trichoderma reesei requires a carrier polypeptide with an intact domain structure. Appl. Env. Microbiol. 69:7073-7082.
Palonen H, M Saloheimo, L Viikari and K Kruus. 2003. Purification, characterization and sequence analysis of a laccase from the ascomycete Mauginiella sp. Enzyme Microb. Technol. 31:403-410.
Paszczynski A, V-B Huynh and R Crawford. 1985. Enzymatic activities of an extracellular Mn-dependent peroxidase from Phanerocahete chrysosporium. FEMS Microbiol Lett 29: 37—41.
Penttila M, H Nevalainen, M Ratto, E Salminen, and J Knowles. 1987. A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene 61:155-164.
Raeder U and P Broda. 1985. Rapid preparation of DNA from filamentous fungi. Lett Appl. MicrobioL 1:17-20.
Rice P, I Longden and A Bleasby. 2000. EMBOSS: The European Molecular Biology Open Software Suite. Trends in Genetics 16:276-277.
Sambrook J, EF Fritsch, and T Maniatis. 1989. Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, New York, US.
Sambrook J and DW RusselL 2001. Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, New York, US.
Schlosser D, R Grey R and W Fritsche. 1997. Patterns of ligninolytic enzymes in Trametes versicolor. Distribution of extra- and intracellular enzyme activities during cultivation on glucose, wheat straw and beech wood, Appl Microbiol Biotechnol 47: 412-418.
Sigoillot C, E Record, V Belle, JL Robert, A Levasseur, P Punt, CAM van der Hondel, A Fourner, JC Sigoillot and M Aster. 2004. Natural and recombinant laccases for pulp and paper bleaching, Appl. Microbiol. BiotechnoL 64: 346-352.
Stone KL, MB Lobresti, ND Williams, JM Crawford, R Deangelis and KR Williams. In Techniques in Protein Chemistry, p. 377-391. T.E. Hugli (ed.). Academic Press, New York 1988.

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CLAIMS
1. A laccase enzyme, characterized in that it is capable by single treatment, in suitable
conditions, of increasing lightness of desized denim at least or above as many units as
sodium hypochlorite.
2. The laccase enzyme according to claim 1, wherein the sodium hypochlorite treatment is
carried out in the presence of 25 ml/1 of 10% sodium hypochlorite (NaOCl) solution for 15
minutes, at the temperature 40"C, at pH above 10.5, in liquor ratio about 1:15, by using
equipment normally used in wet processes in textile industry, such as washing machines.
3. The laccase enzyme according to claim 1 or 2, wherein the laccase treatment is carried out in
the presence of one or more mediators.
4. The laccase enzyme according to any one of claims 1 to 3, wherein the enzyme is obtainable
from a microbial source.
5. The laccase enzyme according to any one of claims 1 to 4, wherein the enzyme is obtainable
from a filamentous fungus.
6. The laccase enzyme according to any one of claims 1 to 5, wherein the enzyme is obtainable
from genus Thielavia.
7. The laccase enzyme according to any one of claims 1 to 6, wherein the enzyme is obtainable
from the deposited strain CBS 116071.
8. The laccase enzyme according to any one of claims 1 to 7, wherein the enzyme comprises
the amino acid sequence SEQ ID NO: 12 or a sequence having at least 74 % identity to SEQ
ID NO: 12.
9. The laccase enzyme according to claim 8, wherein the enzyme lacks the signal sequence.
10. The laccase enzyme according to claim 8 or 9, wherein the enzyme lacks the tail.
11. The laccase enzyme according to any one of the preceding claims, wherein the enzyme is
encoded by the sequence in pALK1342 deposited in E. coli RF5473 under the number DSM
15484.
12. The enzyme according to any one of the preceding claims, wherein the enzyme functions at
pH 3.5 to 8, preferably at pH 4 to 7.5, most preferably at pH 5 to 7.

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13. The enzyme according to any one of the preceding claims, wherein the fabric is desized and
cellulase treated.
14. The enzyme according to any one of the preceding claims, wherein the enzyme is effective in
denim treatment at temperatures 30 to 80 "C, preferably at temperature 50 to 70°C.
15. The enzyme according to any one of the preceding claims, wherein the enzyme is most
effective in denim treatment at temperatures 60 to 70°C.
16. The laccase enzyme according to any one of the preceding claims, wherein the laccase
treatment of the fabric does not cause any essential strength loss.
17. The laccase enzyme according to any one of the preceding claims, wherein the laccase
treatment of the fabric does not cause the loss of abraded look.
18. The laccase enzyme according to any one of the preceding claims, wherein the laccase is
produced in a heterologous production host
19. The laccase enzyme according to any one of the preceding claims, wherein the laccase is
produced in a microbial host
20. The laccase enzyme according to any one of the preceding claims, wherein the laccase is
produced in a filamentous fungus host
21. The laccase enzyme according to any one of the preceding claims, wherein the laccase is
produced in a host of the genus Trichoderma or Aspergillus.
22. A laccase enzyme, characterizedin that it comprises the amino acid sequence SEQ ED
NO: 12 or a sequence showing at least 74 % identity to the sequence SEQ ID NO: 12.
23. The laccase enzyme according to claim 22, wherein the enzyme lacks the signal sequence.
24. The laccase enzyme according to claim 22 or 23, wherein the enzyme lacks the tail.
25. The laccase enzyme according to any one of claims 22 to 24, wherein the enzyme is
obtainable from a microbiai source.
26. The laccase enzyme according to any one of claims 22 to 25, wherein the enzyme is
obtainable from a filamentous fungus.
27. The laccase enzyme according to any one of claims 22 to 26, wherein the enzyme is
obtainable from genus Tfiielavia.

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28. The laccase enzyme according to any one of claims 22 to 27, wherein the enzyme is
obtainable from the deposited strain CBS 116071.
29. The laccase enzyme according to any one of claims 22 to 28, wherein the enzyme is encoded
by the sequence in pALK1342 deposited in E. coli RF5473 under the number DSM 15484.
30. The enzyme according to any one claims 22 to 29, wherein the enzyme functions at pH 3.5
to 8, preferably at pH 4 to 7.5, most preferably at pH 5 to 7.
31. The enzyme according to any one of claims 22 to 30, wherein the pH optimum of the enzyme
is about pH 5.5
32. The laccase enzyme according to any one of claims 22 to 31, wherein the enzyme is
produced in a heterologous production host
33. The laccase enzyme according to any one of claims 22 to 32, wherein the laccase is produced
in a microbial host.
34. The laccase enzyme according to any one of claims 22 to 33, wherein the laccase is produced
in a filamentous fungus host
35. The laccase enzyme according to any one of claims 22 to 34, wherein the enzyme is
produced in a host of the genus Trichoderma or Aspergillus.
36. The laccase enzyme according to any one of claims 22 to 35, wherein the enzyme is capable
by single treatment, in suitable conditions, of increasing lightness of desized denim at least or
above as many units as denim treated by sodium hypochlorite.
37. The laccase enzyme according to claim 36, wherein the sodium hypochlorite treatment is
carried out in the presence of 25 ml/1 of 10% sodium hypochlorite (NaOCl) solution for 15
minutes, at the temperature 40*C, at pH above 10.5, in liquor ratio about 1:15, by using
equipment normally used in wet processes in textile industry, such as washing machine.
38. The laccase enzyme according to claim 36 or 37, wherein the enzyme treatment is carried out
at temperature 30 to 80 *C, preferably at temperature 50 to 70 'C, more preferably at
temperature 60 to 70 "C.
39. The laccase enzyme according to any one of claims 36 to 38, wherein the laccase treatment is
carried out in the presence of one or more mediators.
40. The laccase enzyme according to any one of claims 36 to 39, wherein the enzyme treatment
of denim does not cause any essential strength loss.

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41. The laccase enzyme according to any one of claims 36 to 40, wherein the enzyme treatment
of denim does not cause the loss of abraded look.
42. The laccase enzyme according any one of claims 22 to 41, wherein the enzyme is effective in
stain removal.
43. The laccase enzyme according any one of claims 22 to 41, wherein the enzyme is capable of
decolorizing dyes.
44. A nucleic acid sequence, characterizedin that it encodes the enzyme as defined in
anyone of claims 1 to 43.
45. A vector, characterizedin that it comprises the nucleic acid sequence according to
claim 44.
46. The vector according to claim 45, wherein the nucleic acid sequence has been operably
linked to expression control sequences allowing expression in prokaryotic or eukaryotic host
cells.
47. A host cell, characterizedin that the nucleic acid sequence according to claim 44 or
the vector according to claim 45 or 46 has been introduced into the host cell.
48. The host cell according to claim 47, wherein the host cell is a microbial host celL
49. The host cell according to claim 47 or 48, wherein the host cell belongs to the genus
Trichodenna or Aspergillus.
50. A process for the production of a polypeptide having laccase activity, comprising the steps of
culturing the host cell according to any one of ciaims 47 to 49, and recovering the
polypeptide.
51. A polypeptide having laccase activity encoded by the nucleic acid sequence according to
claim 44, or by the vector according to claim 45 or 46 and which is obtainable by the process
according to claim 50.
52. A process for obtaining an enzyme preparation comprising a polypeptide according to claim
51 comprising the steps of culturing a host cell according to any one of claims 47 to 49 and
either recovering the polypeptide from the cells or separating the cells from the culture
medium and obtaining the supernatant
53. An enzyme preparation obtainable by the process according to claim 52.

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54. An enzyme preparation, which comprises the laccase enzyme according to any one of claims
1 to 43.
55. The enzyme preparation according to claim 53 or 54, wherein the enzyme preparation
comprises a suitable additive(s) selected from the group of stabilizers, buffers, mediators and
preservatives.
56. The enzyme preparation according to any one of claims 53 to 55, wherein the enzyme
preparation is the spent culture medium of the production host.
57. The enzyme preparation according to any one of claims 53 to 56, wherein the enzyme
preparation is in the form of liquid, powder or granulate.
58. A method for treating denim, which comprises contacting denim in an aqueous medium with
a laccase enzyme according to any one of claims 1 to 43 or with an enzyme preparation
according to any one of claims 53 to 57 under suitable conditions for the function of the
enzyme.
59. The method according to claim 58, wherein the treatment is carried out in the presence of a
mediator.
60. The method according to claim 58 or 59 wherein the treatment is carried out at the
temperature of 30 to 80°C, preferably 50 to 70 °C, more preferably at the temperature of 60
to 70°C.
61. The method according to any one of claims 58 to 60, wherein the treatment is carried out at
pH range from pH 3.5 to 8, preferably at pH 4 to 7.5, most preferably at pH 5 to 7.
62. The method according to any one of claims 58 to 61, wherein the treatment is carried out in
15 minutes to 2 hours, preferably in 30 minutes to 90 minutes, more preferably in 30 minutes
to 60 minutes.
63. The method according to any one of claims 58 to 62, wherein the dosage used in the
treatment is 2 to 500, preferably 20 to 200 nkat, most preferably 20 to 100 meat/ g fabric.
64. The method according to any one of claims 58 to 63, wherein the treatment is repeated one or
more times.
65. A method for stain removal, which comprises contacting material to be treated with a laccase
enzyme according to any one of claims 1 to 43 or with an enzyme preparation according to
any one of claims claim 53 to 57 under suitable conditions for the function of the enzyme.

WO 2006/032723 PCT7FI2005/000397
53
66. A method of bleaching pulp, which comprises the step of contacting said pulp with a laccase
enzyme according to any one of claims 1 to 43 or with an enzyme preparation according to
any one of claims 53 to 57 under suitable conditions for the function of the enzyme.
67. A method for treating natural or man-made fibre, which comprises contacting fibre with a
laccase enzyme according to any one of claims 1 to 43 or with an enzyme preparation
according to any one of claims 53 to 57 under suitable conditions for the function of the
enzyme.
68. A method for treating lignocellulolytic fibre, which comprises contacting fibre with a laccase
enzyme according to any one of claims 1 to 43 or with an enzyme preparation according to
any one of claims 53 to 57 under suitable conditions for the function of the enzyme.
69. A method for treating wool, which comprises contacting wool with a laccase enzyme
according to any one of claims 1 to 43 or with an enzyme preparation according to any one
of claims 53 to 57 under suitable conditions for the function of the enzyme.
70. A method for treating hair, which comprises contacting hair with a laccase enzyme
according to any one of claims 1 to 43 or with an enzyme preparation according to any one
of claims 53 to 57 under suitable conditions for the function of the enzyme.
71. A method for treating dye house effluents, which comprises contacting dye house effluents
with a laccase enzyme according to any one of claims 1 to 43 or with an enzyme preparation
according to any one of claims 53 to 57 under suitable conditions for the function of the
enzyme.
72. A method for decolorizing of dyes, which comprises contacting dyes or dye containing
material with a laccase enzyme according to any one of claims 1 to 43 or with an enzyme
preparation according to any one of claims 53 to 57 under suitable conditions for the function
of the enzyme.
73. Use of a laccase enzyme according to of any one of claims 1 to 43 or an enzyme preparation
according to any one of claims claim 53 to 57 for denim treatment, for removing stain, for
bleaching of pulp, for treating of fibers, for treating of wool, for treating of hair, for treating
of dye house effluents or for decolorization of dyes or dye containing material.

The present invention relates to a novel laccase enzyme obtainable from the strains of genus Thielavia. The invention relates also to the nucleic acid sequence encoding the enzyme, a recombinant host into which the nucleic acid sequence has been introduced and a method for the production of the enzyme in a recombinant host. The enzyme of the invention is suitable for several applications, in particular for increasing the lightness of denim.





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Patent Number 269163
Indian Patent Application Number 758/KOLNP/2007
PG Journal Number 41/2015
Publication Date 09-Oct-2015
Grant Date 06-Oct-2015
Date of Filing 01-Mar-2007
Name of Patentee AB ENZYMES OY
Applicant Address TYKKIMAENTIE 15, FI-05200,RAJAMAKI
Inventors:
# Inventor's Name Inventor's Address
1 VALTAKARI, LEENA PINNINTIE 24, FI-05200, RAJAMAKI
2 PALOHEIMO, MARJA RIEKKOPOLKU 5, FI-01450, VANTAA
3 PURANEN, TERHI ORASTIE 5, FI-01900, NURMIJARVI
4 KRUUS, KRISTIINA KAITALAHDENRANTA 7, FI-02260, ESPOO
5 KALLIO, JARNO MUORINPOLKU 8 A 12, FI-04400, JARVENPAA
6 MANTYLA, ARJA AINO ACKTEN TIE 10 A 2, FI-00400, HELSINKI
7 FAGERSTROM, RICHARD KIELOTIE 18 A, FI-02260, ESPOO
8 OJAPALO, PENTTI DAALINTIE 202, FI-04360, TUUSULA
9 VEHMAANPERA, JARI LEPSAMANTIE 359, FI-01800, KLAUKKALA
PCT International Classification Number C12N 9/02
PCT International Application Number PCT/FI2005/000397
PCT International Filing date 2005-09-21
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 20041220 2004-09-21 U.S.A.
2 60/611,819 2004-09-21 U.S.A.