AU725639B2 - DNA sequences, expression of these DNA sequences, thermophilic laccases encoded by the DNA sequences, and the use thereof - Google Patents

Description

WO 98/55628 PCT/EP98/03343 DNA sequences, expression of these DNA sequences, thermophilic laccases encoded by the DNA sequences, and the use thereof The invention relates to DNA sequences which code for proteins having laccase activity, to the expression of these DNA sequences, to the thermophilic laccases encoded by the DNA sequences, and to the use thereof.

A class of enzymes which is of great interest for industrial applications is the class of laccase enzymes (p-hydroxyphenol oxidase, EC 1.10.3.2.).

Laccases are proteins belonging to the family called the "blue copper proteins" and, as a rule, contain four copper ions which are disposed in three copper centers designated type 1 to type 3. Laccases are distinguished by generally being secreted proteins and by possibly having a glycosylation content, from 10 to 45% of the molecular weight. Laccases have a very broad substrate specificity for aromatic compounds which they oxidize.

The electrons generated in this oxidation are used to reduce oxygen. This results in water. The function of laccases, which occur in white rot fungi, is, inter alia, to break down lignin. This is also the source of the interest in employing laccases in paper manufacture for delignification of pulp.

Besides the depolymerization of macromolecular compounds such as lignin, laccases are also able to catalyze the polymerization in particular of aromatic compounds. An example of this is the biosynthesis of lignin in plants in which the laccases occurring in plants are involved. Possible industrial applications of laccases therefore also emerge generally for polymerization reactions of all types, for example in waste water treatment. The use of laccases in organic chemical synthesis is also known, for example in coupling reactions or side-chain oxidation of aromatic compounds. However, for many of these potential 2 applications it is a disadvantage that most of the known laccases are mesophilic, that is to say they have a low temperature optimum and a limited thermal stability.

A precondition for industrial use of laccase enzymes is that they can be prepared at reasonable cost. This is generally possible only by the use of enzymes prepared by recombinant techniques. Various prokaryotic and eukaryotic expression systems are available for protein production. Examples of prokaryotic expression systems are Escherichia coli and Bacillus subtilis. Eukaryotic expression systems which are widely used are cell culture systems both of mammalian cells and of insect cells, and eukaryotic microorganisms such as yeasts or filamentous fungi.

WO 96/00290 describes five laccase genes from filamentous fungus of the subclass of Basidiomycetes, Polyporus pinsitus. One of these laccase genes (LCC1) was prepared as recombinant protein. The thermophilic properties of this enzyme were not investigated further, but the described use of the LCC1 laccase for purposes of hair dyeing suggests that this enzyme has .mesophilic characteristics.

The preparation of a recombinant laccase with 25 thermophilic properties from the filamentous fungus of the subclass of Deuteromycetes, Scytalidium thermo- S"philum, is described in WO 95/33837. It is not known whether this enzyme is suitable for pulp bleaching.

As yet, no process for preparing a thermophilic 30 laccase from a filamentous fungus of the subclass of Basidiomycetes as recombinant protein has been described.

CA: AN 96-203142 discloses various characteristics of a thermophilic laccase. No DNA or protein sequence of this protein is disclosed.

The invention relates to an isolated DNA sequence which codes for a protein having laccase activity, which S.comprises DNA sequence SEQ ID NO: 1 from position 76 up to and including position 1572 or SEQ ID NO: 2 from 3 position 76 up to and including position 1572 or a DNA sequence having a sequence homology of more than with said DNA sequences.

SEQ ID NO: 1 and SEQ ID NO: 2 represent from position 1 to position 75 the DNA sequence coding for the signal sequence for secretion of the protein. This signal sequence can be replaced by any other signal sequences for protein secretion.

The novel DNA sequence can be obtained, for example, by cloning from the Basidiomycetes strain Trametes versicolor TV-1 (deposited at the DSMZ, Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH, D-38124 Braunschweig, under the number DSM 11523) For this purpose, a gene bank is set up from Trametes versicolor TV-1 by methods known per se.

This may be a cDNA or a genomic gene bank.

To isolate the novel DNA sequence in the gene bank, DNA probes which contain laccase-specific DNA sequences are used. DNA probes of this type can be obtained, for example, by means of PCR reaction using DNA primers from genomic DNA from Trametes versicolor TV-1.

The primers used are degenerate DNA sections with a length of, preferably, 14 to 27 bp, whose sequence is established by comparison with sequences of known laccase genes.

The DNA sections suitable as primers are preferably obtained by oligonucleotide synthesis of the DNA sections which have been established.

A novel laccase gene can be isolated, for example, as described in Examples 1 to A laccase gene isolated by way of example in this way can be modified by techniques known to the skilled worker, for example of site directed mutagenesis, at any desired position in the sequence. The invention therefore also embraces a DNA sequence coding for a protein having laccase activity comprising a DNA sequence with a sequence homology of more than 80% with N the DNA sequence SEQ ID NO: 1 from position 76 up to 4 and including position 1572 or SEQ ID NO: 2 from position 76 up to and including position 1572.

To express the novel DNA, the latter is cloned in an expression vector in a manner known per se, and this expression vector containing the laccase gene is introduced into a microorganism and expressed in the microorganism.

The expression vector can be a DNA construct which is integrated into the genome of the host organism and is replicated together with the latter.

Alternatively, the expression vector may be an autonomously replicating DNA construct which does not integrate into the host genome, such as, for example, a plasmid, an artificial chromosome or a comparable extrachromosomal genetic element.

A suitable expression vector ought preferably to contain the following genetic elements: A promoter which promotes expression of the laccase gene in the host organism. This should preferably be a strong promoterrin order to be able to ensure high expression efficiency. The promoter is preferably functionally linked to the 5' end of the laccase gene.

Suitable and preferred promoters are selected from the group of tac promoter, subtilisin promoter, GAL promoter, TAKA amylase promoter, polyhedrin promoter, glucoamylase promoter, gapDH promoter and alcohol oxidase promoter.

Suitable and preferred for expression in E. coli is the tac promoter, for expression in Bacillus is the subtilisin promoter, for expression in the yeast Saccharomyces cerevisiae is the GAL promoter, for expression in Aspergillus niger is the TAKA amylase promoter, for expression in baculovirus-infected insect cells is the polyhedrin promoter, or the glucoamylase promoter from Aspergillus niger or the alcohol oxidase promoter from the yeast Pichia pastoris.

Particularly suitable for expression of the novel thermophilic laccases is the glucoamylase promoter from Aspergillus niger or the alcohol oxidase promoter from the yeast Pichia pastoris.

The expression vector should also preferably contain signals, appropriate for the host organism, for transcription termination and, in eukaryotes, additionally signals for polyadenylation, which should be functionally linked to the 3' end of the laccase gene.

The expressed protein should preferably be secreted by the host organism to the culture medium.

Secretion by the host organism is mediated by an N-terminal signal sequence. The signal sequence is the natural signal sequence present in the laccase gene or is a heterologous signal sequence whose coding DNA is functionally linked to the 5' end of the laccase gene in the expression vector.

The signal sequences of the following secreted proteins are preferred: alpha-cyclodextrin glucosyltransferase from Klebsiella oxytoca, subtilisin from Bacillus subtilis, alpha-factor from Saccharomyces cerevisiae, acid phosphatase from Pichia pastoris, alpha-amylase from Aspergillus niger, glucoamylase from Aspergillus niger or from Aspergillus awamori or the signal sequence naturally present in the laccase gene.

Particularly suitable are the signal sequence naturally present in the laccase gene, and the following heterologous signal sequences: the signal sequence of glucoamylase from Aspergillus niger, or from Aspergillus awamori, the signal sequence of the alpha-factor from Saccharomyces cerevisiae or of the acid phosphatase from Pichia pastoris.

The secretion of the laccase can also be achieved by expression of a fusion protein, where a gene for a secreted protein or for a secreted fragment of this protein is functionally linked to the laccase gene in the expression vector. Particularly preferred in this connection is expression of a fusion protein consisting of an N-terminal fragment of the glucoamylase from Aspergillus niger and the thermo- VDhilic laccase.

6 The point of linkage between the glucoamylase and the laccase is moreover preferably chosen so that the amino acid sequence of this point of linkage serves as recognition site for a processing peptidase in the secretory apparatus of the host cell, the expressed fusion protein being cleaved in vivo and the laccase being released.

The expression vector preferably also contains the gene for a selection marker. The selection markers encoded by the gene can either confer resistance to an antibiotic on the host organism or complement a defect in the host organism.

Preferred selection markers are genes which confer resistance to antibiotics such as ampicillin, kanamycin, chloramphenicol, tetracycline, hygromycin, zeocin or bialaphos. Preferred selection markers for complementation of growth defects are genes such as amdS, pyrG, trpC, His4, niaD, argB, or hygB.

Particularly suitable as selection markers are amdS and the pyrG gene and the His4 gene and the resistance gene for the antibiotic zeocin.

The selection marker gene can moreover be present together with the laccase gene in one DNA molecule or the two genes can be present separately in different DNA molecules. In the latter case, the host organism is cotransformed with both DNA molecules together.

The invention thus also relates to expression vectors which comprise the novel DNA sequences.

Suitable and preferred microorganisms for expressing the novel expression vectors are those of bacterial origin such as E. coli or Bacillus subtilis, microorganisms of eukaryotic origin such as yeasts of the genus Saccharomyces or Pichia, or else filamentous fungi of the genus Aspergillus, Trichoderma, Neurospora or Schyzophillum or eukaryotic cell cultures such as, for example, baculovirus-infected insect cells.

Particularly suitable are filamentous fungi of the genus Aspergillus such as Aspergillus niger or 7 Aspergillus awamori or yeasts such as Saccharomyces cerevisiae or Pichia pastoris.

The proteins encoded by the novel DNA have the following biochemical properties: The proteins have the enzymatic activity of laccases. The pH optimum of the enzyme activity is in the acidic range and is maximal at pH 2.0, with halfmaximal enzyme activity at pH 4.0. The enzyme stability at 45°C and a pH of 4.5 to 6.0 is 100% for a period of up to 2 h.

The enzyme stability at 45°C and a pH of 3.0 is for a period of up to 1 h. The temperature optimum at a pH of 4.5 is 700C. The enzyme activity at 65 0 C is still 80% of the maximal activity. The enzyme activity at 50 800C is still 50% of the maximal activity. The enzyme stability at a pH of 4.5 and temperatures up to 55 0 C is 90% for a period of up to 2 h. The enzyme stability at a pH of 4.5 and a temperature of 65 0 C is 50% for a period of up to 1 h.

The novel isolated proteins comprise the protein sequence SEQ ID NO: 3.

j The novel protein is preferably prepared by expressing novel DNA sequences in an abovementioned microorganism.

25 The DNA is preferably expressed using one of the abovementioned expression vectors in the microorganism.

The invention thus also relates to microorganisms which comprise novel DNA sequences or novel 30 expression vectors.

It is particularly preferable to use combinations of microorganisms and expression systems which also allow secretion of the protein from the microorganism. Examples of such preferred combinations are: Use of the glucoamylase promoter for expressing the novel DNA sequences in Aspergillus niger or Aspergillus awamori. The secretion signal preferably used in this expression system is the signal sequence -8 of the thermophilic laccase itself or the glucoamylase portion of the glucoamylase-laccase fusion protein.

Use of the alcohol oxidase promoter for expressing the novel DNA sequences in Pichia pastoris.

The secretion signal used in this expression system is preferably the signal sequence of the thermophilic laccase itself or the signal sequence of the alphafactor from Saccharomyces cerevisiae or of the signal sequence of the acid phosphatase from Pichia pastoris.

The novel proteins are suitable for all applications known for laccases. They are particularly suitable for the delignification of pulp and the depolymerization of high molecular weight aggregates.

Laccases are further used in the deinking of wastepaper, the polymerization of aromatic compounds in waste water treatment, in this connection particularly of lignin-containing waste waters from pulp bleaching, or in a widened application in the detoxification of contaminated soils. Further areas of use relate to the oxidation of dyes and the activation of dyes by reaction with precursor components to form pigments.

Areas of use in organic synthesis comprise coupling reactions of aromatic compounds or oxidation of aromatic substituents, in this connection for example oxidation of benzyl alcohols to the corresponding aldehydes avoiding further oxidation to the carboxylic acid. In the abovementioned uses, the laccase can be employed on its own in the relevant reactions or else by combination with a reaction-promoting mediator.

Examples of such mediators are ABTS or N-hydroxybenzotriazole.

The invention thus also relates to the use of a novel protein for the delignification of pulp, the depolymerization of high molecular weight aggregates, the deinking of wastepaper, the polymerization of aromatic compounds in waste waters, particularly of lignin-containing waste waters from pulp bleaching, the oxidation of dyes, or the activation of dyes to form ents, the use in organic syntheRAsis for coupling ents, the use in organic synthesis for coupling -o J.

9 reactions of aromatic compounds or the oxidation of aromatic side chains.

The abovementioned uses can be carried out by employing the novel laccase proteins on their own or else by combination with a reaction-promoting mediator.

Fig. 1 shows the structure of the DNA vector pANlaclS.

Fig. 2 shows the structure of the DNA vector pANlac2S.

Fig. 3 shows the structure of the DNA vector pL512.

Fig. 4 shows the structure of the DNA vector pL532.

Fig. 5 shows the dependence of the activity of a novel laccase on the pH.

Fig. 6 shows the pH stability of the novel laccase.

Fig. 7 shows the dependence of the activity of a novel laccase on the temperature.

Fig. 8 shows the temperature stability of the novel laccase.

The following examples serve to illustrate the invention further. The standard methods used in the examples for treating DNA or RNA, such as treatment with restriction endonucleases, DNA polymerases, reverse transcriptase etc., and the standard methods such as transformation of bacteria, Southern and Northern analysis, DNA sequencing, radiolabeling, screening and PCR technology were, unless indicated otherwise, carried out as recommended by the manufacturer of the kits used or, if no manufacturer's instructions were available, in accordance with the prior art known from standard textbooks.

1st example: Production of a cDNA bank from Trametes versicolor TV-1 The strain Trametes versicolor TV-1 was used.

Mycelium from Trametes versicolor was first obtained by culturing on malt-agar plates malt extract, 0.3% 10 peptone from soya bean meal, 1.5% agar-agar, pH 5.0) at 280C for 7 days. Three pieces were punched out of the malt-agar plates and used to inoculate 100 ml of sterile malt extract medium malt extract, 0.3% peptone from soybean meal, pH 5.0) in 500 ml Erlenmeyer flasks. The culture was incubated at 28 0 C, shaking at 100 rpm, for 7 days. The mycelium suspension prepared in this way was filtered with suction through a porcelain funnel, washing with 0.9% saline, and the mycelium was frozen in liquid nitrogen and comminuted with pestle and mortar. RNA was isolated using an RNeasy kit (Qiagen). The yield from 200 mg of mycelium was 100 ig of RNA.

600 jig of RNA were used to isolate mRNA. This took place by chromatography on oligo-dT Sepharose (mRNA isolation kit, Pharmacia). The yield of mRNA was 26 Ag. 7.25 ig of the isolated mRNA were used to synthesize cDNA. A cDNA synthesis kit from Stratagene was used for this. After fractionation, the cDNA was fractionated by agarose gel electrophoresis into a 0.8 2.1 kb size range and a 2.1 5 kb size range. The cDNA of both fractions was isolated from the agarose (Qiagen gel extraction kit) and used to produce the cDNA bank. The cDNA bank was produced in lambda phages (Stratagene, ZAP Express cloning system).

4 x 10 s phages/Ag of vector DNA were obtained from the 0.8 2.1 kb fraction. 1 x 10 5 phages/jig of vector DNA were obtained from the 2.1 5 kb fraction. The resulting phages were amplified by infecting the E. coli strain XL-1 Blue MRF' (Stratagene).

2nd example Production of a chromosomal gene bank from Trametes versicolor Mycelium from Trametes versicolor TV-1 was prepared as described in the 1st example. The mycelium was filtered off with suction through a porcelain funnel and washed with 0.9% saline, then frozen in liquid nitrogen, comminuted with mortar and pestle and 11 divided into 1 g portions. Each 1 g portion of the crushed mycelium was taken up in a sterile sample vessel and immediately mixed with 5 ml of extraction solution (0.1 M tris-HCl, pH 8.0, 0.1 M EDTA, 0.25 M NaCl, 0.6 mg/ml proteinase K) and 0.5 ml of a sodium lauroylsarcosine solution. After incubation at 50 0 C for at least 2 h, the mixture was mixed with 0.85 ml of 5 M NaCl and 0.7 ml of a CTAB solution in 0.7 M NaCl and incubated at 650C for 30 min. After addition of 7 ml of a chloroform/ isoamyl alcohol mixture the mixture was shaken and the two phases were separated by centrifugation.

The aqueous phase was removed and chromosomal DNA was precipitated by adding 0.6 part by volume of isopropanol. The precipitated DNA was subsequently purified on a column (Qiagen Genomic Tip). It was possible in this way to isolate 0.5 mg of chromosomal DNA from 16 g of mycelium.

To produce the chromosomal gene bank, 90 jg of chromosomal DNA from Trametes versicolor TV-1 were completely cut with Eco RI and fractionated by agarose gel electrophoresis. The chromosomal DNA fragments were isolated in the 2 4 kb and 4 kb 10 kb size range and in each case cloned into lambda phages (Stratagene, ZAP Express cloning system). 1 x 10 5 phages/pg of vector DNA were obtained from the 2 4 kb DNA fraction, and 5.4 x 104 phages/pg of vector DNA were obtained from the 4 10 kb DNA fraction. The phages were amplified by infecting the E. coli strain XL-1 Blue MRF'.

3rd example Preparation of a laccase-specific DNA probe from genomic DNA from Trametes versicolor A DNA probe for isolating laccase genes was produced by PCR amplification with degenerate primers from T. versicolor genomic DNA. The degenerate primers were constructed on the basis of a comparison of Z sequences of known laccase genes. The amino acid 1 sequences of the laccase genes, contained in the EMBL 12 gene data bank, from Neurospora crassa, Coriolus hirsutus, Phlebia radiata, Agaricus bisporus and a filamentous fungus which was not characterized in detail from the subclass of Basidiomycetes were compared. It was possible through the comparison of sequences to identify four peptides with a length of to 7 amino acids which were completely conserved in all the laccases. These peptides were translated back to DNA, taking account of degenerate codons, in order to prepare degenerate primers. The primers had the following sequences: A: 5'-TGGCAYGGNTTYTTYCA-3' (SEQ ID NO: 4) B: 5'-TCDATRTGRCARTG-3' (SEQ ID NO: C: 5'-ATTCAGGGATCCTGGTAYCAYWSNCAY-3' (SEQ ID NO: 6) D: 5'-ATACGAGGATCCRTGNCCRTGNARRTG-3' (SEQ ID NO: 7) Primers C and D contained at the 5' end a Bam HI cleavage site (underlined) and, attached thereto, in each case the appropriate degenerate laccase sequence.

Genomic DNA from T. versicolor was isolated from the mycelium of a shaken flask culture as described in the 2nd example. PCR amplifications were carried out in accordance with the prior art familiar to the skilled worker. In a first PCR reaction, 200 ng of chromosomal T. versicolor DNA were employed in a 100 Al PCR reaction which additionally contained 1.25 U of Taq polymerase, 1.25 mM MgC1 2 0.2 mM of each of the four dNTPs and in each case 100 pmol of primers A and B. The other conditions for the specific amplification of the required PCR product were: 5 min at 94°C followed by 7 cycles of 0.5 min at 940C, 1 min at 40 0

C

and 2.5 min at 600C, and 30 cycles of 0.5 min at 94°C, 1 min at 50°C and 2.5 min at 72 0 C. 1 pl from the first PCR reaction was employed in a second PCR reaction which additionally contained 1.25 U of Taq polymerase, 1.25 mM MgC1 2 0.2 mM of each of the four dNTPs and, in S31 each case, 100 pmol of primers C and D. The further 13 conditions for the specific amplication of the required PCR product were: 5 min at 940C, followed by 7 cycles of 0.5 min at 940C, 1 min at 40 0 C and 2.5 min at 600C, and 30 cycles of 0.5 min at 94 0 C, 1 min at 500C and 2.5 min at 720C. A PCR product of about 1.1 kb was obtained. The PCR product was purified by agarose gel electrophoresis, cut with the restriction enzyme Bam HI, and cloned into a pUC18 vector cut with Bam HI and transformed into E. coli. The plasmid was isolated from cultivation of transformed E. coli. DNA sequence analysis of the 5' and 3' ends confirmed that the cloned DNA fragment was the fragment of a laccase gene.

To prepare the DNA probe for screening laccase genes, the laccase-specific PCR fragment was cut out by treatment with Bam HI, isolated by agarose electrophoresis and radiolabeled with a-[ 32 P]-dATP (random priming kit, Boehringer Mannheim). Free radioactivity was removed by chromaiography on Sephadex (Pharmacia). The specific activity of the radiolabeled DNA probe was 1 x 107 cpm/pg of DNA.

4th example Isolation of the cDNA gene of a laccase from Trametes versicolor TV-1 The cDNA gene bank from Trametes versicolor TV-1 described in the 1st example was used. Screening for laccase cDNA genes was carried out in accordance with the prior art. In a first screening round, cells of the E. coli XL-1 Blue MRF' were first cultivated on 10 Petri dishes and then infected with 50,000 phages of the cDNA bank (0.8 2.1 kb fraction, see 1st example) per Petri dish. After incubation at 37 0 C overnight, the newly formed phages were transferred to nylon filters (Stratagene). The filters were then hybridized in accordance with the manufacturer's instructions with the radiolabeled laccase-specific probe (see 3rd example). The hybridization temperature was 450C in a hybridization buffer containing 50% formamide.

7 2 Positive clones were picked and purified by repetition 14 of the screening process. After three rounds of isolation, 20 strongly hybridizing phage clones were isolated in this screening and were recloned by in vivo excision in accordance with a protocol of the manufacturer (Stratagene) into the pBK CMV vector (Stratagene). Analysis of the clones by digestion with restriction endonucleases and DNA sequencing revealed that two laccase genes which were virtually identical at the DNA level had been isolated. Complete DNA sequencing of the two clones revealed that they were alleles of a laccase gene identical in amino acid sequence. The two laccase cDNA genes were called and Lac5.6. Correspondingly, the plasmids with the two laccase cDNA genes were called pLac5.5 and pLac5.6.

example Isolation of the chromosomal gene of a laccase from Trametes versicolor TV-1 Screening for chromosomal laccase genes in the chromosomal gene bank, described in the 2nd example, from Trametes versicolor TV-1 (2 10 kb fraction, see 2nd example) took place in analogy to the screening for cDNA clones described in the 4th example. Once again, the radiolabeled laccase-specific probe described in the 3rd example was used. The hybridization temperature was 45 0 C in a hybridization buffer containing formamide. In the screening, three strongly hybridizing phage clones were isolated and were recloned by in vivo excision in accordance with a protocol of the manufacturer (Stratagene) into the pBK CMV vector (Stratagene). Analysis with restriction endonucleases led to the conclusion that all three clones were identical and had a length of about 7 kb. Analysis by DNA sequencing revealed that all three clones corresponded to the chromosomal gene of the laccase Lac5.6. The coding region of a clone and, in each case, about 1 kb of flanking sequence in the 5' and 3' zregions were sequenced. (SEQ ID NO: 8).

15 6th example Preparation of DNA constructs for the expression of laccase Lac5.5 in Aspergillus cDNA of Trametes versicolor laccase Lac5.5 was functionally linked to expression signals which are specific for filamentous fungi from the Aspergillus family. The following gene expression elements from Aspergillus were used: a) The promoter for the glucoamylase gene (glaA) from Aspergillus niger Verdoes, P.J. Punt, J.M.

Schrickx, H.W. van Verseveld, A.H. Stouthamer and C.A.M.J.J. van den Hondel Transgenic Research 2 (1993), 84 92).

b) The glaA promoter followed by a DNA fragment which codes for the signal sequence and a fragment of mature glucoamylase. Linked to this was a DNA sequence which codes for the cleavage site of the KEX2 protease Broekhuijsen, I.E. Mattern, R. Contreras, J.R. Kinghorn, C.A.M.J.J. van den Hondel (1993), J. Biotechnol. 31, 135-145).

c) The transcription terminator of the trpC gene from Aspergillus nidulans Mullaney, J.E. Hamer, M.M. Yelton and W.E. Timberlake (1985), Mol. Gen.

Genet. 199, 37-45).

However, for functional linkage of the Lac5.5 cDNA to the Aspergillus expression signals, it was first necessary to modify the 5' and 3' regions of the cDNA gene.

A: Linkage of the laccase Lac5.5 cDNA to the glaA promoter: For further processing, the Lac5.5 cDNA gene was recloned into the vector pUC19. To this end, the cDNA gene was isolated as 1.9 kb Eco RI-Xba I fragment from the pBK CMV vector obtained in the 1st example and was subcloned into the pUC19 vector which had previously been cut with Eco RI and Xba I.

The resulting 4.6 kb plasmid was called 16 To modify the start ATG codon of the cDNA gene, primers E and F were used.

Primer E: 5'-CCGGAATTCATGACTGGGCTGCGTCTCCTTCCTTCCTTC-3' (SEQ ID NO: 9) Primer F: 5'-GAGAGGCCCGGGAGCCTGG-3' (SEQ ID NO: Underlining in primer E indicates a Bsp HI cleavage site, and in primer F indicates an Sma I or Xma I cleavage site.

To modify the 3' region of the Lac5.5 cDNA gene, primers G and H were used.

Primer G: 5' -GCTGAATTCGAAGACATCCCCGACACCAAGG-3' (SEQ ID NO: 11) Primer H: 5'-TGCTCTAGAAAGCTTAAGTTCACTGGTCGTCAGCGTCGAGGG-3' (SEQ ID NO: 12) Underlining in primer G indicates a Bbs I cleavage site, and in primer H indicates an Afl II cleavage site.

Primers E and F were used to amplify a fragment 188 bp in size in the 5' region of the Lac5.5 cDNA gene by a PCR reaction. Primers G and H were used to amplify a fragment 110 bp in size in the 3' region of the cDNA gene. A 100 il PCR mixture contained in each case 10 ng of Lac5.5 cDNA (in the pBK CMV vector), U of Tth polymerase, 1.25 mM MgCl 2 0.2 mM of each of the four dNTPs and 140 pmol of each of the pair of primers E and F or of the pair of primers G and H. The PCR reaction was carried out under the following conditions: 5 min at 94°C followed by 30 cycles of 1 min at 940C, 2 min at 50 0 C and 1 min at 72 0 C and finally 7 min at 72 0

C.

The DNA fragment from the PCR reaction with primers E and F was first cut with Eco RI and Xma I and then purified by gel electrophoresis and cloned into Js^ Zihe vector pLac5 which had previously been cut with Eco R and Xma I. This resulted in the vector pLac51 in OFFo a m 17 which a Bsp HI cleavage site has been introduced on the ATG translation start codon.

The DNA fragment from the PCR reaction with primers G and H was first cut with Eco RI and Xba I and then purified by gel electrophoresis and cloned into a pUC19 vector which had previously been cut with Eco RI and Xba I. The insert about 100 bp in size was cut out of the resulting plasmid pLT5 with Bbs I and Xba I. The Bbs I Xba I fragment was finally cloned into the vector pLac51 which had been cut with Bbs I and Xba I.

This resulted in the vector pLac513 which contained a new Afl II cleavage site at the 3' end of the laccase cDNA gene.

The cDNA gene for the Trametes versicolor laccase Lac5.5 with the modified 5' and 3' regions was isolated by partial digestion of the plasmid pLac513 with Bsp HI. This resulted in a 2.6 kb fragment which contained the coding region of the Lac3.5 cDNA gene and about 1.1 kb of the pUC19 vector. This fragment was cut in a second step with Afl II, and the resulting cDNA fragment 1.5 kb in size was isolated. This fragment was ligated into the Afl II Nco I fragment 7.4 kb in size from the vector pAN52-12 which contained a fragment 4.0 kb in size of the glaA promoter from Aspergillus niger, a fragment 0.7 kb in size of the trpC transcription terminator from Aspergillus nidulans and the fragment 2.7 kb in size of the pUC18 vector.

The resulting vector 8.8 kb in size was called pANlacl.

In pANlacl, the glaA promoter region was functionally linked via an Nco I Bsp HI intersection to the ATG translation start codon of the laccase cDNA gene B: Linkage of the laccase Lac5.5 cDNA to the glaA promoter by replacement of the N-terminal signal sequence by a glucoamylase fragment To modify the region of the cDNA gene coding for the N terminus of mature laccase Lac5.5, primers I and F were used.

F1S 18 Primer I: CGAC-3' (SEQ ID NO:13) Underlining in primer I indicates an Eco RV cleavage site.

To modify the 3' region of the Lac5.5 cDNA gene, primers G and H were used (see section A of this example).

Primers I and F were used to amplify a fragment 110 bp in size in the 5' region of the Lac5.5 cDNA gene by a PCR reaction. Primers G and H were used to amplify a fragment 110 bp in size in the 3' region of the cDNA gene. The PCR reactions were carried out as described in section A of this example.

The DNA fragment from the PCR reaction with primers I and F was cut with Eco RI and Xma I and then purified by gel electrophoresis and cloned into the vector pLac5 which had previously been cut with Eco RI and Xma I. This resulted in the vector pLac52 in which there had been inserted, in the 5' region in front of the codon for the first amino acid of the mature laccase protein, an Eco RV cleavage site followed by the codons for the amino acids of the sequence Ile Ser Lys Arg (SEQ ID NO:14). This sequence is a recognition site for KEX2 protease. The 3' region of the laccase cDNA gene in the vector pLac52 was modified as described for the vector pLac51. The insert about 100 bp in size from the PCR reaction with primers G and H was cut out of the plasmid pLT5 with Bbs I and Xba I and was isolated. The Bbs I Xba I fragment was finally cloned into the vector pLac52 which had been cut with Bbs I and Xba I. This resulted in the vector pLac523 which contained a new Afl II cleavage site at the 3' end of the laccase cDNA gene.

The cDNA gene for the Trametes versicolor laccase Lac5.5 with the modified 5' and 3' regions was obtained by cutting the plasmid pLac523 with Eco RV and Afl II, and isolating the resulting Lac5.5 cDNA 19 fragment 1.5 kb in size after agarose gel electrophoresis. This fragment was ligated into the Afl II Eco RV fragment 9.3 kb in size of the vector pAN56-9. Vector pAN56-9 contained a fragment 4.0 kb in size of the glaA promoter from Aspergillus niger, followed by a fragment 2.0 kb in size which codes for a fragment of the Aspergillus niger glaA glucoamylase, a short DNA section which codes for four amino acids of the KEX2 cleavage site, a fragment 0.7 kb in size of the trpC transcription terminator from Aspergillus nidulans and the fragment 2.7 kb in size of the pUC18 vector. The Eco RV and Afl II cleavage sites were arranged 3' of the KEX2 cleavage site. Transformation into E. coli resulted in a vector 10.8 kb in size which was called pANlac2. In pANlac2, the coding region of the cDNA gene for the mature laccase Lac5.5 was linked to the coding region of the glucoamylase gene in such a way that, on expression, firstly a fusion protein consisting of a fragment of the glucoamylase including signal sequence, of the recognition sequence for the KEX2 protease and of the complete laccase Lac5.5 was produced. On secretion, this fusion protein is cleaved by the KEX2 protease, and the mature laccase is secreted into the culture supernatant.

C: Incorporation of the amdS and pyrG selection markers into the vectors pANlacl and pANlac2 pANlacl and pANlac2 were linearized by digestion of 5 pg of each of the vectors with Not I overnight. This was followed by treatment with calf intestinal alkaline phosphatase, phenol/chloroform extraction and ethanol precipitation. The genes for the selection markers amdS (acetamidase) and pyrG decarboxylase) were present on the plasmid pAN52-11 from which they could be isolated as a fragment 6.4 kb in size after digestion with Not I. 0.2 [ig of each of the linearized and phosphatase-treated vectors pANlacl and pANlac2 were Sligated to 0.6 pig of the isolated Not I fragment, and 20 E.coli JM109 was transformed therewith. The plasmid DNA was prepared from ampicillin-resistant colonies from these transformations and was cut with Not I, and analyzed by agarose gel electrophoresis. 6 of 8 analyzed clones from the transformation with the vector pANlacl and 5 of 7 analyzed clones from the transformation with the vector pANlac2 contained the 6.4 kb gene Not I fragment. The vectors equipped with the selection marker genes were called pANlaclS (size 15.3 kb, Fig. 1) and pANlac2S (size 17.2 kb, Fig. 3 of the 6 resulting pANlaclS clones contained the Not I fragment in the required orientation indicated in Fig. 1, and 1 of the 5 resulting pANlac2S clones contained the Not I fragment in the required orientation indicated in Fig.2.

7th example Transformation of Aspergillus The strains of Aspergillus niger AB1.13 (pyrG-) van Hartingsveldt, I.E. Mattern, C.M.J. van Zeijl, P.H. Pouwels and C.A.M.J.J. van den Hondel (1987) Mol.

Gen. Genet. 206, 71 75) and Aspergillus awamori (strain ATCC 11358) were used for the transformation.

The Aspergillus transformation was carried out in accordance with the prior art J. Punt and C.A.J.J.

van den Hondel (1992), Meth. Enzymology 216, 447-457).

Aspergillus protoplasts were obtained by treating the mycelium with Novozym 234. Mycelium from one flask was suspended in 15 ml of a freshly prepared and sterile-filtered solution of the lytic enzyme mixture Novozym 234 (Novo Nordisk) in OM medium (0.27 M calcium chloride, 0.6 M NaCl) in a sterile Erlenmeyer flask. The mycelium resuspended in the enzyme solution was incubated at 30 0 C at slow speed (80 rpm) for 1 to 3 h. During the incubation, the formation of the protoplasts was observed under the microscope. Freely movable protoplasts were normally seen after 1 h. After a large number of freely movable protoplasts had been obtained, they were separated from the remaining 21 mycelium by filtration through Miracloth (Calbiochem) in a glass filter and carefully washed with STC medium (1.2 M sorbitol, 50 mM calcium chloride, 35 mM NaCl, mM tris-HCl, pH Protoplasts were isolated by centrifugation of the suspension in a sterile sample vessel (2000 rpm, 4 0 C, 10 min) and washed twice with STC medium. The protoplast concentration was determined under the microscope in a counting chamber. The protoplast suspension was adjusted to a concentration of 1 x 108 protoplasts/ml for experiments for protoplast regeneration or for transformations.

Protoplasts from Aspergillus niger and Aspergillus awamori were transformed with the plasmids pANlaclS and pANlac2S. Both plasmids contained the pyrG gene (codes for orotidine-5'-phosphate decarboxylase) and the amdS gene (codes for acetamidase) as selection markers. 0.1 ml aliquots of the protoplasts were mixed with 10 Jig of plasmid DNA in incubation vessels with a volume of 12 ml and incubated on ice for 25 min. Then 1.25 ml of a 60% PEG4000 solution (60% PEG4000, 50 mM calcium chloride, 10 mM tris-HCl, pH 7.5) were slowly added with repeated mixing to the transformation mixture. After incubation at 20 0 C for a further 20 min, the reaction vessels were filled with STC medium, mixed and centrifuged at 4 0 C for 10 min. The pellets were resuspended and plated out on selective medium osmotically stabilized with sorbitol. The plates were incubated at 30 0 C for 7 days and checked for growth of colonies. The transformation rate on several experiments was 1 5 transformants/tg of plasmid DNA for Aspergillus niger, and 0.1 0.5 transformants/ug plasmid DNA for Aspergillus awamori.

Transformants were purified by transfer to selective plates with acetamide. Transformants with a high copy number were identified by plating on selective plates with acrylamide. Acrylamide is, in contrast to acetamide, a poor substrate for the acetamidase enzyme encoded by the amdS gene and can assist growth only with transformants having a high 22 copy number of the amdS gene. Transformants capable of functional expression of the laccase enzyme were identified by first plating them out on plates with maltodextrin, an inducer of the expression of the glaA promoter, and, after growth at 28 0 C for 2 days, covered with ABTS agar ABTS, 1% agarose, in McIllvaine buffer, pH Laccase-expressing transformants were indicated by formation of a green halo. Spore suspensions were prepared from transformants which showed both good growth on acrylamide plates and a positive reaction in the activity test with ABTS.

8th example Expression of the Trametes versicolor laccase Lac5.5 in Aspergillus Expression of laccase Lac5.5 in Aspergillus was investigated on the shaken flask scale. The following culture medium was used: a 5% solution of maltodextrin in tap water was autoclaved for 20 min.

Then 1 ml of 1 M Mg sulfate, 0.5 ml of 1000 x trace element solution, 10 ml of 50 x Asp A solution and 5 ml of 10% casamino acids were added to 500 ml of this basic medium. The 1000 x trace element solution had the following composition: 2.2 g of ZnS0 4 x 7H 2 0, 1.1 g of H 3

BO

3 0.5 g of MnC1 2 x 4H 2 0, 0.5 g of FeS0 4 x 7H20, 0.17 g of CoC1 2 x 5H 2 0, 0.16 g of CuS0 4 x 5H 2 0, 0.15 g of Na 2 MoO 4 x 2H 2 0 and 5 g of EDTA were dissolved in 80 ml of H 2 0. The 50 x AspA solution had the composition: 150 g of NaNO 3 13 g of KC1, 38 g of KH 2

PO

4 were dissolved in 500 ml of H 2 0, pH 5.5 adjusted with M KOH. CuS0 4 x 5H 2 0 was also added to the medium in a final concentration of 0.5 mM.

For expression experiments, 50 ml of the culture medium in a 300 ml Erlenmeyer flask were inoculated with 1 x 106 spores/ml. Cultivation took place in a shaking incubator at 30 0 C and 300 rpm.

Samples were taken each day for one week, and the laccase activity in the culture supernatant was deter- 3 mined. The maximum laccase activity was observed with 23 growth between 60 and 100 h, and was between 0.5 and U/ml. The laccase activity was determined by colorimetry with the substrate ABTS (0.1 mM in the assay) in McIllvaine buffer pH 4.5 and at 37 0

C.

McIllvaine buffer was prepared by titrating a 0.1 M citric acid solution against 0.2 M Na 2

HPO

4 solution to pH 4.5. The increase in extinction at 420 nm was measured (extinction coefficient of ABTS at 420 nm: 3.6 x 104 1 x mol I x cm- 1 1 U of laccase activity was defined as 1 Amol of ABTS substrate converted per min.

9th example Expression of the Trametes versicolor laccase Lac5.5 in Pichia pastoris An expression system commercially obtainable from Invitrogen was used with the relevant expression vectors (pPIC3 and pPIC9) and Pichia pastoris strains (GS115 and KM71). The Pichia pastoris strains GS115 and KM71 are histidine-auxotrophic. The expression vectors contained the promoter and terminator of the alcohol oxidase gene AOX1 from Pichia pastoris. The cDNA gene of Trametes versicolor laccase Lac5.5 was cloned between these two genetic regulatory elements. The vector pPIC9 contained, located downstream of the AOX1 promoter, a DNA sequence which codes for the signal sequence of the alpha-factor protein from Saccharomyces cerevisiae, followed by a short DNA section which codes for the recognition sequence Glu-Lys-Arg-Glu-Ala-Glu- Ala (SEQ ID NO: 15). The vectors contained the ampicillin-resistance gene for selection in E. coli.

The vectors contained the HIS4 gene from Pichia pastoris for selection in Pichia pastoris.

A: Construction of a laccase Lac5.5 expression vector with the signal sequence of laccase The plasmid pLac5.5 and primers K and L were used for amplification of the laccase gene. Primers F SUS and G had the following sequence: 9kIC 24 Primer K: -ACTCGAGAATTCACCATGACTGGGCTGCGTCTTCTTCC-3' (SEQ ID NO: 16) Primer L: 5'-ACTAGAGCGGCCGCCTATCACTGGTCGTCAGCGTCGAGGGC-3' (SEQ ID NO: 17) Primer K contained the sequence for an Eco RI cleavage site (underlined) followed by the DNA sequence for the first seven amino acids of the laccase signal sequence. Primer L contained the sequence for a Not I cleavage site (underlined) followed by, in complementary and reverse orientation, the translation stop codon and the DNA sequence for the last 7 amino acids of the laccase PCR amplifications were carried out in accordance with the prior art familiar to the skilled worker. 20 ng of pLac5.5 DNA were employed in a 50 il PCR reaction which additionally contained 0.5 U of Vent polymerase, 1 mM MgC12, 0.2 mM of each of the four dNTPs and in each case 100 pmol of primers K and L. The other conditions for the specific amplification of the required PCR product were: 5 min at 940C followed by cycles of 1 min at 940C, 1 min at 55°C and 2 min at 72°C. The expected PCR fragment with a size of 1.5 kb was obtained. The PCR fragment was purified by agarose gel electrophoresis, cut with the restriction enzymes Eco RI and Not I, precipitated with ethanol and taken up in H20. The vector pPIC3 was cut with Eco RI and Not I, purified by agarose gel electrophoresis, isolated and treated with alkaline phosphatase. This was followed by extraction with phenol/chloroform (ratio 3:1) and an ethanol precipitation. The DNA fragment of the laccase Lac5.5 prepared by PCR amplification was ligated into the pPIC3 vector prepared in this way, and E. coli Top 10F' cells (Invitrogen) were transformed therewith. The plasmid DNA was isolated from ampicillin-resistant clones, and the cloned 1.5 kb insert was identified by restriction S digestion with Eco RI and Not I. 9 of 12 investigated

F-

I- i m ^,FiC7l^ 25 clones were positive. The vector obtained in this way was given the name pL512 (Fig. 3).

B: Construction of a laccase Lac5.5 expression vector with the signal sequence of the alpha-factor from Saccharomyces cerevisiae The plasmid pLac5.5 and primers L and M were used to amplify the laccase gene. Primer M had the following sequence: Primer M: 5'-ACTCGAGAATTCGGGATCGGGCCTGTGCTCGACCTCACG-3' (SEQ ID NO: 18) Primer M contained the sequence for an Eco RI cleavage site (underlined) followed by the DNA sequence for the first nine amino acids of the presumed Nterminus of the processed laccase Lac5.5. The N terminus of the processed laccase Lac5.5 had been deduced from comparison with other laccase sequences.

The DNA fragment for the processed form of the laccase Lac5.5 was prepared as described in section A of this example by PCR amplification with pLac5.5 cDNA and primers L and M. The vector pPIC9 was cut with Eco RI and Not I and prepared like the pPIC3 vector described in section A of this example, and was ligated to the DNA fragment of the laccase Lac5.5 prepared by PCR amplification, and E. coli Top 10F' cells (Invitrogen) were transformed therewith. The plasmid DNA was isolated from ampicillin-resistant clones, and the cloned 1.5 kb insert was identified by restriction digestion with Eco RI and Not I. 3 of 12 investigated clones were positive. The vector obtained in this way was given the name pL532 (Fig. 4).

C: Transformation of Pichia pastoris Pichia pastoris strains GS115 and KM71 were first cultivated in 5 ml of YPD medium yeast extract, 2% peptone, 2% dextrose) at 30 0 C overnight.

S0.2 ml portions of this preculture were used to 26 inoculate two main cultures each of 250 ml of YPD medium, and cultivation was again carried out at 300C overnight until the optical density (OD600 nm) was 1.3 1.5. The yeast cells from a 250 ml main culture were then centrifuged down (1500 x g for 5 min), washed twice with 200 ml of H 2 0 each time and once with 10 ml of 1 M sorbitol and finally taken up in 0.5 ml of 1 M sorbitol.

Plasmid DNA of the vectors pL512 or pL532 were linearized either with Stu I or with Nsi I, precipitated with ethanol and taken up in H 2 0 in a concentration of 1 gg of DNA per il. A transformation mixture contained 80 pl of Pichia pastoris cells and 10 Ag of linearized vector DNA. Transformation took place by electroporation at 1500 V, 25 AF and 200 Ohm (BioRad Gene Pulser). The discharge time was about 4.2 msec.

1 ml of 1 M sorbitol was added to the transformation mixture, which was incubated on ice for 30 min and then plated out in 0.3 ml aliquots on MD plates without histidine (1.34% YNB, yeast nitrogen base, 4 x 10-5% biotin, 1% dextrose, 1.5% agar). Transformants appeared after incubation at 30°C for 3 5 days. Transformants were purified by streaking twice on MD plates. Laccase producers were identified on MM indicator plates. MM indicator plates contained 1.34% YNB, 4 x 10-5% biotin, methanol, 1.5% agar, 1 mM ABTS and 0.1 mM Cu sulfate. The inducer methanol was placed in the lid of the Petri dishes and was renewed each day in order to ensure the colonies were supplied with methanol by diffusion. Laccase producers generated a green halo after incubation at 30 0 C for 2 3 days.

E: Expression in shaken flasks: ml of BMGY medium yeast extract, 2% peptone, 0.1 M K phosphate, pH 6.0, 1.34% YNB, 4 x biotin, 1% glycerol) were inoculated with a laccaseproducing Pichia pastoris transformant and cultivated on a shaker at 300 rpm and 280C for 48 h. The cells Sfrom this preculture were isolated by centrifugation 27 (1500 x g for 10 min) and suspended in 10 ml of main culture medium (MMY, 1% yeast extract, 2% peptone, 1.34% YNB, 4 x 10-% biotin, 3% methanol). MMY medium was supplemented with 0.5 mM Cu sulfate, and the main culture was cultivated further on a shaker at 300 rpm and room temperature. The main culture was supplemented with methanol (0.3 ml/10 ml of medium) at intervals of 24 h. Production of the recombinant laccase started after 24 h. Production rates of up to 4 U/ml were reached 190 h after starting the main culture.

example Isolation of recombinant laccase Recombinant laccase Lac5.5 was obtained by culturing transformed Aspergillus strains in shaken flasks as described in the 8th example. Culture supernatants containing laccase Lac5.5 were concentrated by cross-flow filtration. Sartocon micro filtration modules (Sartorius) with an exclusion' limit of 30 kD were used for this. Concentrated laccase was then lyophilized and dissolved in 20 mM Na phosphate, pH 6.0. The activity of the concentrated recombinant laccase Lac5.5 was 18.6 U/ml.

Laccase concentrated by cross-flow filtration was dialyzed against 20 mM bistris-HCl, pH 6.5. A conductivity of 1.5 mS/cm was measured after this.

Dialyzed laccase was then chromatographed on a column of DEAE-Sepharose (Pharmacia), equilibrated in 20 mM bistris-HCl (loading buffer), pH 6.5. Under these conditions, the laccase binds to the DEAE-Sepharose. On elution with a linear gradient from 0 to 0.5 M NaC1 in loading buffer, the laccase activity was recovered at a salt concentration of 0.15 M NaC1. The laccase fraction from the DEAE-Sepharose column was mixed with ammonium sulfate to 20% saturation and with Na acetate, pH (final concentration 20 mM), and the pH was adjusted to with acetic acid. The laccase fraction prepared in this way was then chromatographed on a column of phenyl-Sepharose (Pharmacia) equilibrated in loading c \^B-irj 28 buffer (20 mM Na acetate, pH 4.5, 20% saturated ammonium sulfate). Under these conditions, the laccase binds to the phenyl-Sepharose. On elution with a linear gradient of 20 0% saturated ammonium sulfate in 20 mM Na acetate, pH 4.5, the laccase activity was recovered at an ammonium sulfate saturation of 16%. The laccase fraction was dialyzed against 20 mM bistris-HCl, pH 6.5, and concentrated by binding to DEAE-Sepharose and subsequent stepped elution with 0.3 M NaC1. Binding and elution with 0.3 M NaCl each took place in 20 mM bistris-HCl, pH 6.5. The yield of laccase activity based on the starting material was 20%. The isolated laccase was analyzed by N-terminal amino acid sequencing. The sequence determined thereby, Gly Ile Gly Pro Val Leu Asp Leu Thr Ile Ser Arg Ala Val (SEQ ID NO: 19), was in agreement with the N terminus of mature laccase Lac5.5 derived from the cDNA sequence and homology comparisons.

llth example Biochemical properties of recombinant laccase The pH and temperature optimum and the pH and temperature stability of recombinant laccase prepared in Aspergillus as described in the 8th example, were investigated. For these experiments, the recombinant laccase Lac5.5 buffer was first changed to McIllvaine buffer, pH 4.5, on Sephadex G25 (Pharmacia, columns).

A: pH optimum: The buffers appropriate for each of the pH values indicated in Fig. 5 were prepared by suitable mixing of unbuffered Na citrate and Na phosphate solutions. The laccase activity of recombinant laccase was then determined at each pH at 370C. As is evident from Fig. 5, the laccase Lac5.5 has an activity 29 optimum in the strongly acidic range for the substrate

-ABTS.

B: pH stability: Laccase Lac5.5 was preincubated in McIllvaine buffer at pH 3.0, 4.5 and 6.0 (temperature 37 0

C).

Aliquots were taken after 0, 30, 60 and 120 min and diluted in McIllvaine buffer, pH 4.5, and the laccase activity was determined at 370C. The laccase activity was not adversely affected by the pretreatment at pH 4.5 and 6.0. The half-life of the laccase at pH was between 60 and 120 min (Fig. 6).

C: Temperature optimum: The laccase activity of the recombinant laccase was determined at the temperatures indicated* in Fig. 7. The laccase activity was determined using the ABTS assay in McIllvaine buffer, pH 4.5. Surprisingly, it was found from this that the temperature optimum of the laccase Lac5.5 is 70 0 C. The temperatures at which the measured laccase activity is still half the maximum were 50 0 C and 80 0

C.

D: Temperature stability: :25 Laccase Lac5.5 was preincubated in McIllvaine buffer, pH 4.5, at 45, 55 and 650C. Aliquots were taken after 0, 30, 60 and 120 min and diluted in McIllvaine buffer, pH 4.5, and the laccase activity was determined at 37 0 C. The laccase activity was not adversely 30 affected by the pretreatment at 45 0 C. Measurement after preincubation at 550C for 120 min showed 80% activity still remaining. The laccase half-life at 65°C was min (Fig. 8).

"Comprises/comprising" when used in this specification is taken to specify the presence. of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, neintegers, steps, components or groups thereof.

30 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Consortium fuer elektrochemische Industrie GmbH STREET: Zielstattstrasse CITY: Munich COUNTRY: Germany POSTAL CODE: D-81379 TELEPHONE: 089 748440 TELEFAX: 089 74844350 (ii) TITLE OF APPLICATION: DNA sequences, of these DNA thermophilic encoded by sequences, an thereof (iii) NUMBER OF SEQUENCES: 19 (iv) COMPUTER-READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (EPO) expression sequences, laccases the DNA d the use INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS LENGTH: 1572 base pairs TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA to mRNA VU" 'S -v -4 31 (iii) HYPOTHETICAL: NO (iii) [sic] ANTISENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Trametes versicolor STRAIN: TV-i (vii) IMMEDIATE SOURCE: CLONE: piacSS (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: ATGACTGGGC TGCGTCTTCT TCCTTCCTTC GCGGCGTTGG CCGTGACCGT GTCTCTCGCG CTCAACGCGT TGGCTGGGAT CGGGCCTGTG CTCGACCTCA CGATCTCCAA CGCGGTGGTG 120 2 0 TCGCCCGATG GCTTCTCTCG CGCGGCGGTC GTTGCGAACG ACCAGGCTCC CGGGCCTCTC 180 ATTACGGGCC AGATGGGCGA CCGTTTCCAG ATCAATGTGG TCAACAAGCT GTCGAACCAC 240 2S ACTATGCTCA AGTCGACCAG CATCCACTGG CACGGCTTCT TCCAGAAGGG TACGAACTGG 300 GCCGATGGCC CCGCGTTCGT GAACCAGTGC CCGATCGCGA CTGGTCACTC GTTCCTTTAC 360 GACTTCCAGG TCCCGGACCA GGCCGGGACG 1TCTGGTACC ACAGCCATT7 GTCTACCCAG 420 TACTGTGACG GGTTGAGAGG TCCTTTCGTC GTCTACGACC CGAACGACCC TCATGCCAGC 480 CTCTACGACG TGGACAACGA TGACACCGTC ATCACCCTCG CTGACTGGTA CCATACCGCT 540 GCCAAGCTTG GGCCGGCCTT CCCTCCTGGC TCTGATGCGA CGTTGATCAA TGGGCTCGGA 600 32 CGTACAGCGG CCACCCCCAA CGCGGATCTC GCTGTCATTA GTGTCACGCA CGGCAAGCGG 660 TACCGTTTCC GCCTGGTGTC GATGTCCTGC GACCCCGCGT ACACGTTCAG CATCGACGAC 720 CACTCGATGA CCATCATCGA GGCGGACTCA GTCAACACAA AGCCGCTCGA GGTCGACTCG 780 1 0 ATCCAGATCT TCGCCGGCCA GCGCTACTCG TTCGTGCTGG AGGCAAACCA GGACGTCGGC 840 AACTATTGGG TCCGCGCGGA CCCGCTGTTT GGCACGACGG GCTTCGATGG GGGTATCAAC 900 TCTGCGATCC TCCGGTACGA CACCGCGTCG CCGACCGAGC CGACCACGAC GCAGGCCACC 960 TCTACGAAGC CGTTGAAGGA GACGGACCTT GAGCCTCTCG CGTCGATGCC GGTGCCTGGC 1020 TCTGCTGTCT CGGGTGGTGT GGACAAGGCG ATTAACTTCG CTTTCAGCTT CAACGGGTCC 1080 AACTTCTTCA TCAACGGCGC GACCTTCCAG CCGCCCACCA CTCCCGTTCT GCTGCAGATC 1140 ATGAGCGGTG CCCAGGCTGC TAGCGACCTC CTCCCGTCCG GTGACGTCTA CGCCCTGCCG 1200 TCGGACTCGA CCATCGAGCT CTCGTTCCCC GCGACTACTG GTGCTCCCGG TGCCCCCCAC 1260 CCCTTCCACT TGCACGGTCA CACCTTCGCC GTTGTGCGCA GCGCGGGCAG CGCTGAGTAC 1320 AACTACGACA ACCCCATCTG GCGCGACGTC GTCAGCACTG GTACCCCTGC AGCGGGCGAT 1380 33 AACGTCACCA TTCGCTTCAG GACTGACAAC CCTGGCCCGT GGTTCCTCCA CTGCCACATC 1440 GACTTCCACT TGGAGGCCGG CTTCGCCGTG GTCATGGCTG AAGACATCCC CGACACCAAG 1500 GCCGACAACC CTGT7CCTCA GGCGTGGTCA GACCTTTGCC CCATCTACGA CGCCCTCGAC 1560 GCTGACGACC AG 1572 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 1572 base pairs TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iii) [sic] ANTISENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Trametes versicolor STRAIN: TV-).

(vii) IMMEDIATE SOURCE: CLONE: plac56 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 ATGACTGGGC TGCGTCTTCT TCCTTCCTTC GCGGCGTTGG CCGTGACCGT GTCGCTCGCG 34 CTCAACGCGT TGGCCGGGAT CGGGCCCGTG CTCGACCTTA CGATCTCCAA TGCGGTTGTT 120 TCGCCCGATG GCTTCTCTCG CGCGGCGGTC GTCGCGMACG ACCAGGCTCC

CGGGCCTCTC

180 ATCACGGGCC AGATGGGCGA CCGCTTCCAG ATCAATGTGG TCAACAAGCT GTCGAACCAC 240 ACCATGCTTA AATCGACCAG CATCCACTGG CACGGCTTCT TCCAGAAGGG CACGAACTGG 300 GCGGACGGCC CTGCGTTCGT GAACCAATGC CCGATTGCGA CGGGCCACTC GTTCCTTTAC 360 GACTTCCAGG TCCCGGACCA GGCCGGGACG TTCTGGTACC ACAGCCATCT GTCTACTCAG 420 TACTGCGATG GCTTGAGGGG TCCGTTCGTC GTCTACGACC CGAATGACCC TCATGCCAGT 480 CTCTACGATG TGGACAACGA TGACACCGTC ATCACCCTCG CCGATTGGTA CCATACTGCT 540 GGCCGGCCTT CCCTCCTGGC TCTGATGCGA CGTTGATCAA TGGGCTCGGA 600 CGTACAGCGG CCACCCCCAA CGCGGACCTC GCTGTCATCA GCGTCACGCA CGGCAAGCGG 660 TACCGTTTCC GCCTGGTGTC GATGTCCTGC GACCCCGCGT ACACCTTCAG CATCGACGAC 720 CACTCGATGA CCATCATCGA GGCGGACTCG GTCAACACGA AGCCGCTCGA GGTCGACTCG 780 ATCCAGATCT TCGCCGGCCA GCGCTACTCG TTCGTGCTGG AGGCAAACCA

GGACGTCGGC

35 AACTATTGGG TCCGCGCGGA CCCGCTGTTT GGCACGACGG GCTTCGATGG GGGTATCAAC 900 TCTGCGATCC TCCGGTACGA CACCGCGTCG CCGACCGAGC CGACCACGAC GCAGGCCACC 960 TCTACGAAGC CGTTGAAGGA GACGGACCTT GAGCCTCTCG CGTCGATGCC GGTGCCTGGC 1020 TCTGCTGTGT CGGGTGGTGT GGACAAGGCG ATTAACTTCG CTTTCAGCTT CAACGGGTCC 1080 AACTTCTTCA TCAACGGCGC GACCTTCCAG CCGCCCACCA CTCCCGTTCT GCTGCAGATC 1140 ATGAGCGGTG CCCAGGCTGC TAGCGACCTC CTCCCGTCCG GTGACGTCTA CGCCCTGCCG 1200 TCGGACTCGA CCATCGAGCT CTCG1TCCCC GCGACTACTG GTGCTCCCGG TGCCCCCCAC 1260 CCCTTCCACT TGCACGGTCA CACCTTCGCC G77GTGCGCA GCGCGGGCAG CGCTGAGTAC 1320 AACTACGACA ACCCCATCTG GCGCGACGTC GTCAGCACTG GTACCCCTGC AGCGGGCGAT 1380 AACGTCACCA 7TCGCTTCAG GACTGACAAC CCTGGCCCGT GGTTCCTCCA CTGCCACATC 1440 GACTTCCACT TGGAGGCCGG CTTCGCCGTG GTCATGGCTG AAGACATCCC CGACACCAAG 1500 GCCGACAACC CTGTTCCTCA GGCGTGGTCA GACCTTTGCC CCATCTACGA CGCCCTCGAC 1560 GCTGACGACC AG 17 1572 36- (2)INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 524 amino acids TYPE: Amino acid TOPOLOGY: Linear (ii) MOLECULE TYPE: Protein (iii) HYPOTHETICAL: YES (vi) ORIGINAL SOURCE: ORGANISM: Trametes versicolor STRAIN: TV-1 (ix) FEATURES: NAME/KEY: Protein LOCATION: 1..524 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Met Thr Gly Leu Arg Leu Leu Pro Ser Phe Ala Ala Leu Ala Val Thr 1 5 10 Val Ser Leu Ala Leu Asn Ala Leu Ala Gly lie Gly Pro Val Leu Asp 20 25 Leu Thr lie Ser Asn Ala Val Val Ser Pro Asp Gly Phe Ser Arg-Ala 40 Ala Val Val Ala Asn Asp Gin Ala Pro Gly Pro Leu lie Thr Gly Gin 55 Met Gly Asp Arg Phe Gin lie Asn Val Val Asn Lys Leu Ser Asn His 70 75 3 5 Thr Met Leu Lys Ser Thr Ser lie His Trp His Gly Phe Phe Gin Lys 90 'u Gly Thr Asn Trp Ala Asp Gly Pro Ala Phe Val Asn Gin Cys Pro lie 100 105 110 -37 Ala Thr Gly His Ser Phe Leu Tyr Asp Phe Gin Val Pro Asp Gin Ala 115 120 125 Gly Thr Phe Trp Tyr His Ser His Leu Ser Thr Gin Tyr Cys Asp Gly 130 135 140 Leu Arg Gly Pro Phe Val Val Tyr Asp Pro Asn Asp Pro His Ala Ser 145 150 155 160 Leu Tyr Asp Val Asp Asn Asp Asp Thr Val lie Thr Leu Ala Asp Trp 165 170 175 Tyr His Thr Ala Ala Lys Leu Gly Pro Ala Phe Pro Pro Gly Ser Asp 180 185 190 Ala Thr Leu lie Asn Gly Leu Gly Arg Thr Ala Ala Thr Pro Asn Ala 195 200 205 2 C Asp Leu Ala Val lie Ser Val Thr His Gly Lys Arg Tyr Arg Phe Arg 210 215 220 Leu Val Ser Met Ser Cys Asp Pro Ala Tyr Thr Phe Ser lie Asp Asp 225 230 235 240 His Ser Met Thr lie lie Glu Ala Asp Ser Val Asn Thr Lys Pro Leu 245 250 255 Glu Val Asp Ser lie Gin lie Phe Ala Gly Gin Arg Tyr Ser Phe Val 260 265 270 Leu Glu Ala Asn Gin Asp Val Gly Asn Tyr Trp Val Arg Ala Asp Pro 275 280 285 Leu Phe Gly Thr Thr Gly Phe Asp Gly Gly lie Asn Ser Ala lie Leu 290 295 300 Arg Tyr Asp Thr Ala Ser Pro Thr Glu Pro Thr Thr Thr Gin Ala Thr 305 310 315 320 38 Ser Thr Lys Pro Leu Lys Glu Thr Asp Leu Glu Pro Leu Ala Ser Met 325 330 335 Pro Val Pro Gly Ser Ala Val Ser Gly Gly Val Asp Lys Ala lie Asn 340 345 350 Phe Ala Phe Ser Phe Asn Gly Ser Asn Phe Phe lie Asn Gly Ala Thr 355 360 365 Phe Gin Pro Pro Thr Thr Pro Val Leu Leu Gin lie Met Ser Gly Ala 370 375 380 Gin Ala Ala Ser Asp Leu Leu Pro Ser Gly Asp Val Tyr Ala Leu Pro 385 390 395 400 Ser Asp Ser Thr lie Glu Leu Ser Phe Pro Ala Thr Thr Gly Ala Pro 405 410 415 Gly Ala Pro His Pro Phe His Leu His Gly His Thr Phe Ala Val Val 420 425 430 Arg Ser Ala Gly Ser Ala Glu Tyr Asn Tyr Asp Asn Pro lie Trp Arg 435 440 445 Asp Val Val Ser Thr Gly Thr Pro Ala Ala Gly Asp Asn Val Thr lie 450 455 460 Arg Phe Arg Thr Asp Asn Pro Gly Pro Trp Phe Leu His Cys His lie 465 470 475 480 Asp Phe His Leu Glu Ala Gly Phe Ala Val Val Met Ala Glu Asp lie 485 490 495 Pro Asp Thr Lys Ala Asp Asn Pro Val Pro Gin Ala Trp Ser Asp Leu 500 505 510 Cys Pro lie Tyr Asp Ala Leu Asp Ala Asp Asp Gin 515 520 39 (2)INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: YES (iii) [sic] ANTISENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: TGGCAYGGNT TYTTYCA 17 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: YES (iii) [sic] ANTISENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TCDATRTGRC ARTG 14 INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: YES (iii) [sic] ANTISENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: ATTCAGGGAT CCTGGTAYCA YWSNCAY INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: YES (iii) [sic] ANTISENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: ATACGAGGAT CCRTGNCCRT GNARRTG -41 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 3284 base pairs TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) [sic] ANTISENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Trametes versicolor STRAIN: TV-l (vii) IMMEDIATE SOURCE: CLONE: plac56chr (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: TTGGAATGGG CCCGCCGGTG TCGATCGCAA ACAGGTGAGT TCAGTACTAG CCCATCAGCG 6 CAGCAGCATT TGCGGCGAAG AAGTGTCCGC CCCACGCTCA TCACCCAGCG CCCTTCTCGA 120 CATCGGAACC GCCGCAACAC AGGGAAGAGG CCATTTCGCC CATCCAAGGC TCGGGGATCT 180 TCTCACGACG CGAGGGCATT TCGCGCGAGC GTCGCAGCCG GTGCGCCGAG GCTGCATGAT 240 CTGTGCGGCT CCTGCGCGTA TGCCGCTCGG GTCGCCGAGA CACAGCGAGA CATCTGCAGC 300 CGGGGCGGCG CGCAACCAGC TCGGCTGTTT GGAGTGCGTC GATGCAACGC GTCGACGTCC 360 42 ATCGGGGACG GCGCGTGGCT TGGCACGCGT AGCACCGACG CGCACTATAA AGGCGATGCG 420 GCAGAGAAGA GGCGGAGCAC CACGTTCAGT CCCTTCCT1G GATTCCGGGC AGCTTACTCC 480 TTrCTCGCCTC TCTCTGCCTC CTTTCCTTCG GGCTTCTACT CTTCTTTTCT ATTTCGCTTC 540 TGTTCGAGGG TAGAACACAG AACACTATGA CTGGGCTGCG TCTTCTTCCT TCCTTCGCGG 600 CGTTGGCCGT GACCGTGTCG CTCGCGCTCA ACGCGTTGGC CGGGATCGGG CCCGTGCTCG 660 ACCTTACGAT CTCCAATGCG GTTGTTTCGC CCGATGGCTT CTCTCGCGCG GCGGTCGTCG 720 CGAACGACCA GGCTCCCGGG CCTCTCATCA CGGGCCAGAT GGGCGACCGC TTCCAGATCA 780 ATGTGGTCAA CAAGCTGTCG AACCACACCA TGCTTAAATC GACCAGCATC GTGAGTATTC 840 AATCTGGGCG TGGGGGTACG GGCTGCACTG ACGCAAGTAC ACGCTTCGCA GCACTGGCAC 900 GGCTTCTTCC AGAAGGGCAC GAACTGGGCG GACGGCCCTG CGTTCGTGAA CCAATGCCCG 960 ATTGCGACGG GCCACTCGTT CCTTTACGAC TTCCAGGTCC CGGACCAGGC CGGTATGTGA 1020 TCACGGAAGG TGTGCAcGAA CCCAGCACTG ACGGTCATGT AGGGACGTTC TGGTACCACA 1080 GCCATCTGTC TACTCAGTAC TGCGATGGCT TGAGGGGTCC GTTCGTCGTC TACGACCCGA S 1140 43 ATGACCCTCA TGCCAGTCTC TACGATGTGG ACAACGGTAA GCAGTTCAGA TTGCGAATCC 1200 TTGGCGGTCT ATTGACATCC CGGCCAGATG ACACCGTCAT CACCCTCGCC GATTGGTACC 1260 ATACTGCTGC CAAGCTTGGG CCGGCCTTCC CGTAAGTTGG ATTGTCAGTC TGTCTGTTCT 1320 CTACTTACTA ATCACGGGCT GCAGTCCTGG CTCTGATGCG ACGTTGATCA ATGGGCTCGG 1380 ACGTACAGCG GCCACCCCCA ACGCGGACCT CGCTGTCATC AGCGTCACGC ACGGCAAGCG 1440 GTAAGAGCGG CTGTACCTTC CTCTTGCTCG CAGCTGCTCA AACTATGG 1TrATAGGTA 1500 CCGTTTCCGC CTGGTGTCGA TGTCCTGCGA CCCCGCGTAC ACCTTCAGCA TCGACGACCA 1560 CTCGATGACC ATCATCGAGG CGGACTCGGT CAACACGAAG CCGCTCGAGG TCGACTCGAT 1620 CCAGATCTTC GCCGGCCAGC GCTACTCGTT CGTGCTGGAG GCAAACCAGG ACGTCGGCAA 1680 CTATTGGGTC CGCGCGGACC CGCTGTTTGG CACGACGGGC TTCGATGGGG GTATCAACTC 1740 TGCGATCCTC CGGTACGACA CGGCGTCGCC GACCGAGCCG ACCACGACGC AGGCCACi:TC 1800 TACGAAGCCG TTGAAGGAGA CGGACCTTGA GCCTCTCGCG TCGATGCCGG TGGTAAGTCT 1860 GACTAGCACT TCATCTTTGA TGGTATGCTC ATGCAACTCT CCAGCCTGGC TCTGCTGTGT 1920 44 CGGGTGGTGT GGACAAGGCG ATTAACTTCG CTTTCAGCTT CGTACGTCCA ACTCACGATT 1980 TCCCCCTTGA GATTTATA11 GATGGCTCCA TTGACGGCTC CTCATAGAAC GGGTCCAACT 2040 TCTTCATCAA CGGCGCGACC TTCCAGCCGC CCACCACTCC CGTTCTGCTG CAGATCATGA 2100 GCGGTGCCCA GGCTGCTAGC GACCTCCTCC CGTCCGGTGA CGTCTACGCC CTGCCGTCGG 2160 ACTCGACCAT CGAGCTCTCG TTCCCCGCGA CTACTGGTGC TCCCGGTGCC CCCCACCCCT 2220 TCCACTTGCA CGGTGTAAGT TGTCATCTCA ATGTTCCGTT TGGGCCCCGA TACTAACGGC 2280 TAGATAGCAC ACCTTCGCCG TTGTGCGCAG CGCGGGCAGC GCTGAGTACA ACTACGACAA 2340 CCCCATCTGG CGCGACGTCG TCAGCACTGG TACCCCTGCA GCGGGCGATA ACGTCACCAT 2400 TCGCTTCAGG GTGAGTTGCT ATCATTATCC CCTCCTGTGT A.ATCGGTCGC TGACAGTCCT 2460 GCAGACTGAC AACCCTGGCC CGTGGTrCCT CCACTGCCAC ATCGACTTCC ACTTGGAGGC 2520 CGGCTTCGCC GTGGTCATGG CTGAAGACAT CCCCGACACC AAGGCCGACA ACCCTGTTCC 2580 TCGTGAGTAT TACCCCCCAA TCCCGTCAAG GCGCGCACTA ACAGGGTATT GCTGCAGAGG 2640 CGTGGTCAGA CCTTTGCCCC ATCTACGACG CCCTCGACGC TGACGACCAG TGAACACGCC 2700 1 45 TCACGAGATC GTCAACCATT TCCTCAATCA TTGACTTACC GACTTGCTAT TTCTAACACG 2760 CTATTTGCGA ACCCCCGCTC TCCCCTCTCT CACACTACGG TCCCTUCGTG AACATGGACT 2820 TGCATGGACT TUGGAUrGTA GAAAGTTTAC ACAGCTGTAT AGTCGAATTA TCCCCGTAAT 2880 GCATGGTAGT GCCGCTGGCC TTTACCTCAA TCA11GTTAT CATGATATGG CCATCATAAA 2940 CATCACTGAC ATCTACTMAT CTGCTGTTAG T1TGGGACC TCAAGAAGAT AAACGCCCGT 3000 CTACCACGAT GTGACGCGCG CGATACGTGA ATGTGACTGA TCGCGTTCCA TTATTCAAAA 3060 CGCGTCGGCT GGCGGCCAGG CCAAG1TGCT CCTCTCTCTC CGACGACGAC CACCCCTGGC 3120 TCTCTTACCC ACCTTCTCTG CACCATGACG GCAGACTACA GACTACAGTC TCTCGACGAT 3180 CCGACGGCGG TCATCCAAGA GCTCTACCGC GCCCATCCAG ACCCGAACGG TTTCCCCCGC 3240 CTCGTTGCTG AGCACTTCCA AAAGCTCTTC GAGAACCGAA CATG 3284 INFORMATION FOR SEQ, ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: YES (iii) [sic] ANTISENSE: NO 46 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: CCGGAATTCA TGACTGGGCT GCGTCTCCTT CCTTCCTTC 39 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: YES (iii) [sic] ANTISENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GAGAGGCCCG GGAGCCTGG 19 INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: YES (iii) [sic] ANTISENSE: NO 47 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GCTGAATTCG AAGACATCCC CGACACCAAG G 31 INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE.TYPE: cDNA (iii) HYPOTHETICAL: YES (iii) [sic] ANTISENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: TGCTCTAGAA AGCTTAAGTT CACTGGTCGT CAGCGTCGAG GG 42 INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 43 Base pairs TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: YES (iii) [sic] ANTISENSE: NO 48 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: CCGGAATTCG ATATCCAAGC GCGGGATCGG GCCTGTGCTC GAC 43 INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: Amino acid TOPOLOGY: Linear (ii) MOLECULE TYPE: Peptide (iii) HYPOTHETICAL: YES FRAGMENT TYPE: Internal (vi) ORIGINAL SOURCE: ORGANISM: Aspergillus niger (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Ile Ser Lys Arg 1 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: Amino acid TOPOLOGY: Linear (ii) MOLECULE TYPE: Peptide (iii) HYPOTHETICAL: YES FRAGMENT TYPE: Internal 49 (vi) ORIGINAL SOURCE: ORGANISM: Saccharomyces cerevisiae (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Glu Lys Arg Glu Ala Glu Ala 1 INFORMATION FOR SEQ ID NO: 16: SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: YES (iii) [sic] ANTISENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: ACTCGAGAAT TCACCATGAC TGGGCTGCGT CTTCTTCC INFORMATION FOR SEQ ID NO: 17: SEQUENCE CHARACTERISTICS: LENGTH: 41 Base pairs TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA %(iii) HYPOTHETICAL: YES 50 (iii) [sic] ANTISENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: ACTAGAGCGG CCGCCTATCA CTGGTCGTCA GCGTCGAGGG C 41 INFORMATION FOR SEQ ID NO: 18: SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: Nucleic acid STRANDEDESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: YES (iii) [sic] ANTISENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: ACTCGAGAAT TCGGGATCGG GCCTGTGCTC GACCTCACG 39 INFORMATION FOR SEQ ID NO: 19: SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids TYPE: Amino acid TOPOLOGY: Linear (ii) MOLECULE TYPE: Peptide (iii) HYPOTHETICAL: NO FRAGMENT TYPE: N terminus

"I

-1- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: Gly Ile Gly Pro Val Leu Asp leu Thr Ile Ser Arg Ala Val 1 5

Claims (9)

1. An isolated DNA sequence which codes for a protein havingi laccase activity, which comprises DNA sequence SEQ ID NO: 1 from position 76 up to and including position 1572 or SEQ ID NO: 2 from position 76 up to and including position 1572 or a DNA sequence having a sequence homology of more than 80% with said DNA sequences.

2. An expression vector which comprises a DNA sequence as claimed in claim 1.

3. An expression vector as claimed in claim 2, which additionally comprises: a promoter which mediates expression of the laccase gene in a host organism, and signals for transcription termination which are appropriate for the host organism and are functionally linked to the 3' end of the DNA sequence as claimed in claim 1.

4. An expression vector as claimed in claim 3, wherein the promoter is selected from the group of tac promoter, subtilisin promoter, GAL promoter, TAKA amylase promoter, polyhedrin promoter, glucoamylase promoter, GAPDH promoter and alcohol oxidase promoter.

An expression vector as claimed in claim 3 or 4, which additionally comprises an N-terminal signal sequence.

6. An expression vector as claimed in claim wherein the N-terminal signal sequence is the natural signal sequence present in the laccase gene, or is 30 selected from the group of signal sequences of the following secreted proteins: alpha-cyclodextrin glucosyltransferase from Klebsiella oxytoca, subtilisin from Bacillus subtilis, alpha-factor from Saccharomyces cerevisiae, acid phosphatase from Pichia pastoris, alpha-amylase from Aspergillus niger or glucoamylase from Aspergillus niger or from Aspergillus awamori. 53

7. An expression vector as claimed in claim wherein a gene for a secreted protein or a gene section for secreted fragment of a protein is functionally linked to the laccase gene.

8. A microorganism strain which comprises an expression vector as claimed in any of claims 2 to 7.

9. An isolated protein comprising the protein sequence SEQ ID NO: 3. The use of the protein as claimed in claim 9 for the delignification of pulp, the depolymerization of high molecular weight aggregates, the deinking of wastepaper, the polymerization of aromatic compounds in waste waters, particularly of lignin-containing waste waters from pulp bleaching, the oxidation of dyes, or the activation of dyes to form pigments, the use in organic synthesis for coupling reactions of aromatic compounds or the oxidation of aromatic side chains. DATED this 22 d day of August 2000 CONSORTIUM FUR ELEKTROCHEMISCHE INDUSTRIE GMBH WATERMARK PATENT AND TRADE MARK ATTORNEYS 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA CJH:VRH P16546AUOO.DOC 0o *oo