EP0994950A2

EP0994950A2 - Dna sequences, expression of said dna sequences, thermopile laccases coded by said dna sequences and the use thereof

Info

Publication number: EP0994950A2
Application number: EP98937446A
Authority: EP
Inventors: Rupert Pfaller; Günter Wich
Original assignee: Consortium fuer Elektrochemische Industrie GmbH
Current assignee: Consortium fuer Elektrochemische Industrie GmbH
Priority date: 1997-06-06
Filing date: 1998-06-04
Publication date: 2000-04-26
Also published as: AU8624698A; CA2292723A1; WO1998055628A2; NO995948D0; WO1998055628A3; CN1259166A; AU725639B2; JP2000513588A; DE19724039A1; PL337465A1; KR20010013482A; NZ501312A; ID23856A; NO995948L; BR9815544A

Abstract

The invention relates to DNA sequences which code for proteins with laccase activity, comprising one of the DNA sequences (SEQ ID NO:1, SEQ ID NO:2). The invention also relates to the expression of these DNA sequences, thermopile laccases coded by said DNA sequences and their use for delignifying cellulose, depolymerizing high-molecular aggregates, de-inking waste paper, polymerising aromatic compounds in waste liquids, especially waste liquids from cellulose bleaching, oxidising colorants and activating colorants to produce pigments, and their use in organic synthesis in coupling reactions of aromatic compounds or the oxidation of aromatic side chains.

Description

DNA sequences, expression of these DNA sequences, thermophilic laccases encoded by the DNA sequences and their use

The invention relates to DNA sequences which code for proteins with laccase activity, the expression of these DNA sequences, the ophilous laccases encoded by the DNA sequences and their use.

An enzyme class that is of great interest for industrial applications is the enzyme class of laccases (p-hydroxyphenol oxidase, EC 1.10.3.2.). Laccases belong to the protein family of the so-called "blue copper proteins" and generally contain four copper ions, which are arranged in three copper centers, type 1 to type 3. Laccases are characterized by the fact that they are generally secreted proteins and that they can contain a glycosylation fraction of 10 to 45% of the molecular weight. Laccases have a very broad substrate specificity for aromatic compounds that oxidize them. The generated during this oxidation

Electrons are used to reduce oxygen. This creates water. The function of the laccases that occur in white rot fungi is, among other things, lignin degradation. Hence the interest to use laccases in papermaking to delignify pulp.

In addition to the depolymerization of macromolecular compounds such as lignin, laccases can also catalyze the polymerization of especially aromatic compounds. An example of this is lignin biosynthesis in plants, in which laccases occurring in plants are involved. Technical applications for laccases therefore generally arise in all types of polymerization reactions, for example in wastewater treatment. The use of laccases is also known in organic chemical synthesis, for example in coupling reactions or the side chain oxidation of aromatics. For many of these potential applications, however, it is disadvantageous that most known laccases are mesophilic, that is, they have a low temperature optimum and limited temperature stability.

A prerequisite for the technical application of laccase enzymes is that they can be produced inexpensively. This is generally only possible through the use of recombinantly produced enzymes. 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 with widespread use are cell culture systems of both mammalian and insect cells as well as eukaryotic microorganisms such as yeasts or filamentous fungi.

WO96 / 00290 describes five laccase genes from the filamentous fungus of the subclass of the Basidiomycetes, Polyporus pinsitus. One of these laccase genes (LCC1) was produced as a recombinant protein. The thermophilic properties of this enzyme have not been investigated further, the use of LCC1 laccase described for hair coloring suggests that this enzyme has a mesophilic character.

The production of a recombinant laccase with thermophilic properties from the filamentous fungus of the subclass of Deuteromycetes, Scytalidiu thermophilum, is described in WO 95/33837. It is not known whether this enzyme is suitable for pulp bleaching.

No process for the production as a recombinant protein of a thermophilic laccase from a filamentous fungus from the subclass of the Basidiomycetes has yet been described.

CA: AN 96-203142 discloses various characteristics of a thermophilic laccase. DNA or protein sequence of this protein are not mentioned. The invention relates to a DNA sequence which codes for a protein with laccase activity, characterized in that it includes the 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 with a sequence homology greater than 80% to said DNA sequences.

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

The DNA sequence according to the invention can be obtained, for example, by cloning from the basidiomycete strain Trametes versicolor TV-1 (deposited with the DSMZ German Collection of Microorganisms and Cell Cultures GmbH, D-38124 Braunschweig under the number DSM 11523). For this purpose, a gene bank of Trametes versicolor TV-1 is created using methods known per se. It can be a cDNA or a genomic library.

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

Degenerate DNA segments with a length of preferably 14 to 27 bp are used as primers, the sequence of which is determined by sequence comparison of known laccase genes.

The DNA segments suitable as primers are preferably obtained by oligonucleotide synthesis of the specified DNA segments. A laccase gene according to the invention can be isolated, for example, as described in Examples 1 to 5.

A laccase gene isolated in this way, for example, can be modified using techniques known to those skilled in the art, such as, for example, site-directed mutagenesis, at the desired position in the sequence. Therefore, a DNA sequence coding for a protein with laccase activity comprising a DNA sequence with a sequence homology greater than 80% to the DNA sequence SEQ ID NO: 1 from position 76 up to and including position 1572 or SEQ ID NO: 2 from position 76 to position 1572 inclusive of the invention.

To express the DNA according to the invention, it is cloned in a manner known per se in an expression vector, 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 that is integrated into the genome of the host organism and replicated together with it. Alternatively, the expression vector can be an autonomously replicating DNA construct that does not integrate into the host genome, e.g. a plasmid, an artificial chromosome or a comparable extrachromosomal genetic element.

A suitable expression vector should preferably contain the following genetic elements:

A promoter that mediates expression of the lacca gene in the host organism. It should preferably be a strong promoter so that high expression performance can be guaranteed. The promoter is preferably operably linked to the 5 'end of the laccase gene. Promoters which are preferably suitable are selected from the group consisting of tac promoter, subtilisin promoter, GAL promoter, TAKA amylase promoter, polyhedrin promoter, glucoamylase promoter, gapDH promoter and alcohol oxidase promoter.

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

The glucoamylase promoter from Aspergillus niger or the alcohol oxidase promoter from the yeast Pichia pastoris are particularly suitable for the expression of the thermophilic laccases according to the invention.

Furthermore, should preferably be suitable for the host organism

Signals for the transcription termination and in eukaryotes additionally contain signals for the polyadenylation in the expression vector and be functionally linked to the 3 'end of the laccase gene.

The expressed protein should preferably be secreted into the growth medium by the host organism. Secretion from the host organism is mediated by an N-terminal signal sequence. The signal sequence is the natural signal sequence contained in the laccase gene or a heterologous signal sequence, the coding DNA of which is functionally linked in the expression vector to the 5 'end of the laccase gene.

The signal sequences of the following secreted proteins are preferred: alpha-cyclodextrin glucosyl transferase from Klebsieila 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 Aspergillus awamori or the signal sequence naturally contained in the laccase gene.

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

The secretion of laccase can also be achieved by the expression of a fusion protein in which 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. Expression of a fusion protein between an N-terminal fragment of the Aspergillus niger glucoamylase and the thermophilic laccase is particularly preferred here.

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

Furthermore, the expression vector preferably contains the gene for a selection marker. The selection markers encoded by the gene can either confer resistance to an antibiotic to 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. As a selection marker for the complementation of growth defects Genes such as amdS, pyrG, trpC, His4, niaD, argB, or hygB are preferred.

The amdS and the pyrG gene as well as the His4 gene and the resistance gene for the antibiotic Zeocin are particularly suitable as selection markers.

The selection marker gene can be contained together with the laccase gene in one DNA molecule or both genes can be contained separately in different DNA molecules. In the latter case, the host organism is co-transformed with both DNA molecules.

The invention thus also relates to expression vectors which contain the DNA sequences according to the invention.

Suitable microorganisms for expression of the expression vectors according to the invention are preferably 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 filamentous fungi of the genus Aspergillus, Trichoderma, Neurospora or Schyzophillum cell culture or eukaryotic cell . B. Baculovirus infected insect cells.

Filamentous mushrooms of the genus Aspergillus such as Aspergillus niger or Aspergillus awamori or yeasts such as Saccharomyces cerevisiae or Pichia pastoris are particularly suitable.

The proteins which are encoded by the DNA according to the invention 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 a maximum of pH 2.0, with half maximum enzyme activity at pH 4.0.

The enzyme stability at 45 ° C and a pH of 4.5 to 6.0 is 100% up to a period of 2 h. The enzyme stability at 45 ° C and a pH of 3.0 is 50% up to a period of 1 h.

The temperature optimum is at a pH of 4.5 70 ° C. From 65 - 75 ° C the enzyme activity is still 80% of the maximum activity.

From 50 - 80 ° C the enzyme activity is still 50% of the maximum activity.

The enzyme stability at a pH of 4.5 and temperatures up to 55 ° C is 90% up to a period of 2 h. The enzyme stability at a pH of 4.5 and a temperature of 65 ° C is 50% up to a period of 1 h.

The proteins according to the invention comprise the protein sequence SEQ ID NO: 3.

The protein according to the invention is preferably produced by expressing DNA sequences according to the invention in a microorganism mentioned above.

The DNA is preferably expressed in the microorganism using one of the expression vectors mentioned above.

The invention thus also relates to microorganisms which contain DNA sequences according to the invention or expression vectors according to the invention.

Combinations of microorganisms and expression systems are particularly preferably used which also enable the protein to be secreted from the microorganism. Examples of such preferred combinations are:

The use of the glucoamylase promoter to express the DNA sequences according to the invention in Aspergillus niger or Aspergillus awamori. The signal sequence of the thermophilic laccase itself or the glucoamylase portion of the glucoamylase-laccase fusion protein is preferably used as the secretion signal in this expression system. The use of the alcohol oxidase promoter to express the DNA sequences according to the invention in Pichia pastoris. The signal sequence of the thermophilic laccase itself or the signal sequence of the alpha factor from Saccharomyces cerevisiae or else the signal sequence of the acidic phosphatase from Pichia pastoris is preferably used as the secretion signal in this expression system.

The proteins according to the invention are suitable for all applications known for laccases. They are particularly suitable for the delignification of pulp or the depolymerization of high-molecular aggregates. Laccases are also used in the deinking of waste paper, the polymerization of aromatic compounds in wastewater treatment, especially lignin-containing wastewater from cellulose bleaching, or in an extended application for the detoxification of contaminated soils. Further areas of application concern the oxidation of dyes or the activation of dyes by reaction with precursor components with pigment formation. Areas of application in organic synthesis include coupling reactions of aromatic compounds or the substituent oxidation of aromatics, here z. B. the oxidation of benzyl alcohols to the corresponding aldehydes while avoiding further oxidation to the carboxylic acid. With the above The laccase can be used on its own in the corresponding reactions or else in combination with a reaction-enhancing mediator. Examples of such mediators are ABTS or N-hydroxybenzotriazole.

The invention thus also relates to the use of a protein according to the invention for the delignification of cellulose, the depolymerization of high molecular aggregates, the deinking of waste paper, the polymerisation of aromatic compounds in waste water, especially lignin-containing waste water from cellulose bleaching, the oxidation of dyes or the Activation of dyes for pigment formation, the use in organic synthesis in coupling reactions of aromatic compounds or the oxidation of aromatic side chains.

The above Uses can be taken on their own by using the laccase proteins according to the invention, or they can be carried out in combination with a reaction-enhancing mediator.

1 shows the structure of the DNA vector pANlaclS.

2 shows the structure of the DNA vector pANlac2S.

3 shows the structure of the DNA vector pL512.

4 shows the structure of the DNA vector pL532.

5 shows the dependence of the activity of a laccase according to the invention on the pH.

6 shows the pH stability of the laccase according to the invention.

7 shows the dependence of the activity of a laccase according to the invention on the temperature.

8 shows the temperature stability of the laccase according to the invention.

The following examples serve to further explain the invention. The standard methods used in the examples for the treatment of DNA or RNA, such as the treatment with restriction endonucleases, DNA polymerases, reverse transcriptase etc. as well as the standard methods such as transformation of bacteria, Southern and Northern analysis, DNA sequencing, radioactive labeling, Unless otherwise specified, screening and PCR technology were performed as recommended by the manufacturer of the kits used, or if none Manufacturer's instructions were available, according to the state of the art known from the standard textbooks.

Example 1: Production of a cDNA bank from Trametes versicolor TV-1

The Trametes versicolor TV-1 strain was used. Trametes versicolor mycelium was first obtained by culturing for 7 days at 28 ° C on malt agar plates (3% malt extract, 0.3% peptone from soybean meal, 1.5% agar agar, pH 5.0). Three pieces were punched out of the malt agar plates and 100 ml of sterile malt extract medium (3% malt extract, 0.3% peptone from soybean meal, pH 5.0) were inoculated in 500 ml Erlenmeyer flasks. The culture was incubated with shaking at 100 rpm for 7 days at 28 ° C. The mycelium suspension prepared in this way was sucked off through a porcelain suction filter, washed with 0.9% saline and the mycelium was frozen in liquid nitrogen and ground with a mortar and pestle. RNS was isolated with an RNeasy kit (Qiagen). The yield from 200 mg mycelium was 100 μg RNA.

600 µg RNA was used to isolate mRNA. This was done by chromatography over Oligo-dT Sepharose (mRNA isolation kit, Pharmacia). The yield of mRNA was 26 μg. 7.25 μg of the isolated mRNA was used to synthesize cDNA. A cDNA synthesis kit from Stratagene was used for this. After separation by agarose gel electrophoresis, the cDNA was fractionated into a size range of 0.8-2.1 kb and a size range of 2.1-5 kb. The cDNA of both fractions was isolated from the agarose (Qiagen gel extraction kit) and used to prepare the cDNA library. The cDNA bank was produced in lambda phages (Stratagene, ZAP Express cloning system). 4 × 10 ⁵ phages / μg vector DNA were obtained from the 0.8-2.1 kb fraction. 1 × 10 phages / μg vector DNA were obtained from the 2.1-5 kb fraction. The phages obtained were amplified by infection of the E. coli strain XL-1 Blue MRF '(Stratagene). 2nd example

Production of a chromosomal gene bank from Trametes versicolor

Trametes versicolor TV-1 mycelium was prepared as described in Example 1. The mycelium was aspirated through a porcelain suction filter and washed with 0.9% saline, then frozen in liquid nitrogen, crushed with a mortar and pestle and divided into 1g portions. 1 g each of the ground mycelium was taken up in a sterile sample vessel and immediately with 5 ml 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 10% (w / v) sodium lauroyl sarcosine solution. After incubation at 50 ° C for at least 2 h, the mixture was mixed with 0.85 ml of 5 M NaCl and 0.7 ml of a 10% (w / v) CTAB solution in 0.7 M NaCl and 30 min at 65 ° C incubated. After adding 7 ml of a chloroform-isoamyl alcohol mixture (24: 1), 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 parts by volume of isopropanol. The precipitated DNA was then purified on a column (Qiagen Genomic Tip). In this way, 0.5 mg of chromosomal DNA could be isolated from 16 g of mycelium.

To produce the chromosomal gene bank, 90 μg of chromosomal DNA from Trametes versicolor TV-1 were completely cut with Eco RI and separated by agarose gel electrophoresis. The chromosomal DNA fragments were isolated in the size range from 2 - 4 kb and from 4 kb - 10 kb and cloned in each case in lambda phages (Stratagene, ZAP Express cloning system). 1 × 10 phages / μg vector DNA were obtained from the 2–4 kb DNA fraction and 5.4 × 10 ⁴ phages / μg vector DNA were obtained from the 4–10 kb DNA fraction. The phages were amplified by infection of the E. coli strain XL-1 Blue MRF '. 3rd example

Preparation of a laccase-specific DNA probe from Trametes versicolor genomic DNA

A DNA probe for isolating laccase genes was generated by PCR amplification from T. versicolor genomic DNA with degenerate primers. The degenerate primers were constructed using a sequence comparison of known laccase genes. The amino acid sequences of the laccase genes from Neurospora crassa, Coriolus hirsutus, Phlebia radiata, Agaricus bisporus and an unspecified filamentous fungus from the subclass of Basidiomycetes contained in the EMBL gene database were compared. The sequence comparison identified four peptides with a length of 5 to 7 amino acids that were completely conserved in all laccases. Taking into account degenerate codons, these peptides were translated back into DNA to produce degenerate primers. The primers had the following sequences:

A 5 '-TGGCAYGGNTTYTTYCA-3' (SEQ ID NO: 4) B 5 '-TCDATRTGRCARTG-3' (SEQ ID NO: 5) C 5 '-ATTCAGGGATCCTGGTAYCAYWSNCAY-3' (SEQ ID NO: 6) D 5 '-ATACGARNGTR -3 '(SEQ ID NO: 7)

Primers C and D contained a Barn HI cleavage site (underlined) at the 5 'end and the corresponding degenerate laccase sequence was connected to them.

Genomic DNA from T. versicolor was extracted from the mycelium

Shake flask culture isolated as described in Example 2. PCR amplifications were carried out in accordance with the prior art known to the person skilled in the art. In a first PCR reaction, 200 ng of chromosomal T. versicolor DNA were used in a 100 μl PCR reaction, which also contained 1.25 U Taq polymerase, 1.25 mM MgCl ₂ , 0.2 mM each of the four dNTPs and each Contained 100 pmol of primers A and B. The further conditions for the specific amplification of the desired PCR Product were: 5 min at 94 ° C, followed by 7 cycles of 0.5 min at 94 ° C, 1 min at 40 ° C and 2.5 min at 60 ° C, and 30 cycles of 0.5 min at 94 ° C, 1 min at 50 ° C and 2.5 min at 72 ° C. 1 μl of the first PCR reaction was used in a second PCR reaction, which also contained 1.25 U Taq polymerase, 1.25 mM MgCl ₂ , 0.2 mM each of the four dNTPs and 100 pmol each of the primers C and D contained. The other conditions for the specific amplification of the desired PCR product were: 5 min at 94 ° C, followed by 7 cycles of 0.5 min at 94 ° C, 1 min at 40 ° C and 2.5 min at 60 ° C as well 30 cycles of 0.5 min at 94 ° C, 1 min at 50 ° C and 2.5 min at 72 ° C. A PCR product of approx. 1.1 kb was obtained. The PCR product was purified by agarose gel electrophoresis, cut with the restriction enzyme Barn HI and cloned into a pUC18 vector cut with Barn HI and E. coli transformed. The plasmid was isolated from the cultivation of transformed E. coli. DNA sequence analysis from the 5 ¹ and 3 'end confirmed that the cloned DNA fragment was the fragment of a laccase gene.

To prepare the DNA probe for the screening of laccase genes, the laccase-specific PCR fragment was cut out by treatment with Bam HI, isolated by agarose electrophoresis and radioactively labeled with α- [32P] -dATP ("Random

Priming "kit, Boehringer Mannheim). Free radioactivity was separated by chromatography on Sephadex G25 (Pharmacia). The specific activity of the radioactively labeled DNA probe was 1 × 10 ⁷ cpm / μg 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 first example was used. The screening for laccase cDNA genes was carried out according to the prior art. In In a first round of screening, cells from E. coli XL-1 Blue MRF 'were first cultured on 10 petri dishes and then infected with 50,000 phages from the cDNA library (0.8-2.1 kb fraction, see 1st example) per petri dish. After incubation at 37 ° C overnight, the newly formed phages were placed on nylon filters

(Stratagene) transferred. The filters were then hybridized with the radioactively labeled laccase-specific probe (see 3rd example) in accordance with the manufacturer's recommendations. The hybridization temperature was 45 ° C. in a hybridization buffer with 50% formamide. Positive clones were picked and purified by repeating the screening procedure. After three rounds of separation, 20 strongly hybridizing phage clones were isolated in the screening and were cloned into the pBK CMV vector (Stratagene) by "in vivo excision" according to a manufacturer's specification (Stratagene). Analysis of the clones by digestion with restriction endonucleases and DNA sequencing revealed that two laccase genes had been isolated, which were almost identical at the DNA level. Complete DNA sequencing of the two clones revealed that they were alleles of a laccase gene, the amino acid sequence of which was identical. The two laccase cDNA genes were named Lac5.5 and Lac5.6. The plasmids with the two laccase cDNA genes pLac5.5 and pLac5.6 were designated accordingly.

5th example

Isolation of the chromosomal gene of a laccase from Trametes versicolor TV-1

The screening for chromosomal laccase genes in the chromosomal library of Trametes versicolor TV-1 described in the 2nd example (2-10 kb fraction, see 2nd example) was carried out analogously to the screening for cDNA clones described in the 4th example. Again, the radioactively labeled laccase-specific probe described in the 3rd example was used. The hybridization temperature was 45 ° C. in a hybridization buffer with 50% formamide. At the screening were three strongly hybridizing phage clones were isolated, which were cloned according to a manufacturer's instructions (Stratagene) by "in vivo excision" 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 approx. 7 kb. Analysis by DNA sequencing showed that all three clones corresponded to the chromosomal gene of the Laccase Lac5.6. The coding region of a clone and in each case approximately 1 kb flanking sequence in the 5 'and in the 3' region were sequenced. (SEQ ID NO: 8)

6th example

Preparation of DNA constructs for the expression of laccase

Lac5.5 in Aspergillus

Trametes versicolor Laccase Lac5.5 cDNA 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 (JC Verdoes, PJ Punt, JM Schrickx, HW van Verseveld, AH Stouthamer and CAMJJ van den Hondel Transgenic Research 2 (1993), 84 - 92). b) The glaA promoter followed by a DNA fragment coding for the signal sequence and a fragment of the mature glucoamylase. This was linked to a DNA sequence which codes for the cleavage site of the KEX2 protease (MP Broekhuij sen, IE Mattern, R. Contreras, JR Kinghorn, CAMJJ van den Hondel (1993), J. Biotechnol. 3., 135-145 ). c) the transcription terminator of the trpC gene from Aspergillus nidulans (E. J. Mullaney, J. E. Hamer, M. M. Yelton and W. E. Timberlake (1985), Mol. Gen. Genet. 19, 37-45). For the functional linkage of the Lac5.5 cDNA with the Aspergillus expression signals, however, it was first necessary to modify the 5 'and 3' regions of the Lac5.5 cDNA gene.

A: Linking the Laccase Lac5.5 cDNA with the glaA promoter: For further processing, the Lac5.5 cDNA gene was cloned into the vector pUC19. For this purpose, the Lac5.5 cDNA gene was isolated as a 1.9 kb Eco RI-Xba I fragment from the pBK CMV vector obtained in Example 1 and subcloned into the pUC19 vector precut with Eco RI and Xba I. The resulting 4.6 kb plasmid was called pLac5.

Primers E and F were used to modify the start ATG codon of the Lac5.5 cDNA gene.

Primer E: 5 '-CCGGAATTCATGACTGGGCTGCGTCTCCTTCCTTCCTTC-3' (SEQ

ID NO: 9)

Primer F: 5 '-GAGAGGCCCGGGAGCCTGG-3' (SEQ ID NO: 10)

Underlined in primer E is an example HI interface, in primer F an Sma I or Xma I interface.

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

Primer G: 5 '-GCTGAATTCGAAGACATCCCCGACACCAAGG-3' (SEQ ID NO: 11) Primer H: 5 '-TGCTCTAGAAAGCTTAAGTTCACTGGTCGTCAGCGTCGAGGG-3' (SEQ ID NO: 12)

Underlined in primer G is a Bbs I interface, in primer H an Afl II interface.

A 188 bp fragment was amplified in the 5 'region of the Lac5.5 cDNA gene with the primers E and F by a PCR reaction. With the primers G and H was in the 3 'range of

Lac5.5 cDNA gene amplified a 110 bp fragment. A 100 μl PCR mixture each contained 10 ng Lac5.5 cDNA (in the pBK CMV vector), 0.5 U Tth polymerase, 1.25 mM MgCl ₂ , 0.2 mM each of the four dNTPs and 140 pmol each of the primer pair E and F, or the primer pair 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 94 ° C., 2 min at 50 ° C. and 1 min at 72 ° C and finally 7 min at 72 ° C. The DNA fragment from the PCR reaction with the primers E and F was first 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 pLac51, in which a Bsp HI interface was introduced at the ATG codon of the translation start.

The DNA fragment from the PCR reaction with the 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 precut with Eco RI and Xba I. The approximately 100 bp insert with Bbs I and Xba I was cut out from the plasmid pLT5 formed in the process. The Bbs I - Xba I fragment was finally cloned into the vector pLac51 cut with Bbs I and Xba I. This created the vector pLac513, which contained a new Afl II 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 Example HI. This resulted in a 2.6 kb fragment which contained the coding region of the Lac5.5 cDNA gene and approximately 1.1 kb of the pUC19 vector. This

Fragment was cut in a second step with Afl II and the resulting 1.5 kb Lac5.5 cDNA fragment was isolated. This fragment was ligated into the 7.4 kb Afl II - Nco I fragment of the vector pAN52-12, which contains a 4.0 kb fragment of the glaA promoter from Aspergillus niger and a 0.7 kb fragment of the trpC transcription terminator from Aspergillus nidulans and the 2.7 kb fragment of the pUC18 vector contained. The resulting 8.8 kb vector was called pANlacl. In pANlacl, the glaA promoter region was functionally linked to the translation start ATG codon of the laccase cDNA gene via an Nco I - Bsp HI crossing. B: Linking of the Laccase Lac5.5 cDNA with the glaA promoter via an exchange of the N-terminal signal sequence for a fragment of the glucoamylase

Primers I and F were used to modify the region of the cDNA gene coding for the N-terminus of the mature laccase Lac5.5.

Primer I: 5 '-CCGGAATTCGATATCCAAGCGCGGGATCGGGCCTGTGCTCGAC-3' (SEQ ID NO: 13)

Underlined in Primer I is an Eco RV interface. Primers G and H were used to modify the 3 'region of the Lac5.5 cDNA gene (see Section A of this example).

With the primers I and F, a 110 bp fragment in the 5 'region of the Lac5.5 cDNA gene was amplified by a PCR reaction. A 110 bp fragment was amplified with the primers G and H in the 3 'region of the Lac5.5 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 first 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 Xmal. This resulted in the vector pLac52, in which an Eco RV interface followed by the codons for the amino acids of the sequence Ile Ser Lys Arg (Seq ID NO: 14) had been introduced in the 5 'region in front of the codon for the first amino acid of the mature laccase protein . This sequence is a recognition site for the KEX2 protease. The 3 'region of the laccase cDNA gene in the vector pLac52 was modified as described for the vector pLac51. The plasmid pLT5 became about 100 bp

Insert from the PCR reaction with the primers G and H with Bbs I and Xba I cut out and isolated. The Bbs I - Xba I fragment was finally in the with Bbs I and Xba I cloned vector pLac52 cloned. This resulted in the vector pLac523, which contained a new Afl II 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 the resulting 1.5 kb Lac5.5 cDNA fragment was isolated after agarose gel electrophoresis. This fragment was ligated into the 9.3 kb Afl II - Eco RV fragment of the vector pAN56-9. Vector pAN56-9 contained a 4.0 kb fragment of the Aspergillus niger glaA promoter, followed by a 2.0 kb fragment coding for a fragment of the Aspergillus niger glaA glucoamylase, a short DNA section for encoded four amino acids of the KEX2 cleavage site, a 0.7 kb fragment of the trpC transcription terminator from Aspergillus nidulans and the 2.7 kb fragment of the pUC18 vector. The Eco RV and Afl II interfaces were arranged 3 'to the KEX2 cleavage site. After transformation into E. coli, a 10.8 kb vector was obtained, 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, when expressed, first a fusion protein from a fragment of the glucoamylase including signal sequence, the recognition sequence for the KEX2 protease and the complete laccase Lac5.5 was created. During the 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 μg of the vectors each night with Not I. This was followed by treatment with alkaline phosphatase from calf intestine, extraction with phenol / chloroform and precipitation with ethanol. The genes for the amdS (acetamidase) and pyrG selection markers (Orotidine-5 '-monophosphate decarboxylase) were contained on the plasmid pAN52-II, from which they could be isolated as a 6.4 kb fragment after digestion with Not I. 0.2 μg each of the linearized and phosphatase-treated vectors pANlacl and pANlac2 were ligated with 0.6 μg of the isolated Not I fragment and thus E. coli JM109 were transformed. The plasmid DNA was prepared from ampicillin-resistant colonies from these transformations, cut with Not I and analyzed by agarose gel electrophoresis. 6 out of 8 clones analyzed from the transformation with the vector pANlacl and 5 out of 7 clones analyzed from the transformation with the vector pANlac2 contained the 6.4 kb Not I fragment. The vectors equipped with the selection marker genes were designated as pANlaclS (size 15.3 kb, Fig. 1) and pANlac2S (size 17.2 kb, Fig. 2). 3 of the 6 pANlaclS clones obtained contained the Not I fragment in the desired orientation shown in FIG. 1, 1 of the 5 pANlac2S clones obtained contained the Not I fragment in the desired orientation shown in FIG. 2.

7th example

Aspergillus transformation

The strains Aspergillus niger ABI.13 (pyrG ^" ) (W. van Hartingsveldt, IE Mattern, CMJ van Zeijl, PH Pouwels and CAMJJ van den Hondel (1987) Mol. Gen. Genet. 206, 71 - 75) were used for the transformation. and Aspergillus awamori (strain ATCC 11358) The transformation of Aspergillus was carried out according to the prior art (PJ Punt and CAJJ van den Hondel (1992), Meth. Enzymology 216, 447-457)

Protoplasts from Aspergillus were obtained by treating the mycelium with Novozyτn 234. In a sterile Erlenmeyer flask, mycelium from a 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) . The mycelium resuspended in the enzyme solution was used at low speed (80 rpm) Incubated for 1 to 3 h at 30 ° C. The formation of protoplasts was observed under the microscope during the incubation. Free moving protoplasts were usually seen after 1 h. After a large number of freely moving protoplasts had been obtained, they were separated from residual 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, 10 mM Tris-HCl, pH 7.5). Protoplasts were isolated by centrifuging the suspension in a sterile sample vessel (2000 rpm, 4 ° C, 10 min) and washed twice with STC medium. The concentration of protoplasts was determined under the microscope in a counting chamber. The protoplast suspension was adjusted to a concentration of 1 x 10 "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 (coding for the orotidine 5'-phosphate decarboxylase) and the amdS gene (coding for acetamidase) as selection markers. In incubation vessels of 12 ml volume, 0.1 ml aliquots of the protoplasts were mixed with 10 μg plasmid DNA and incubated on ice for 25 min. Thereafter, 1.25 ml of a 60% PEG4000 solution (60% PEG4000, 50 mM calcium chloride, 10 mM

Tris-HCl, pH 7.5) was added to the transformation mixture. After a further 20 min incubation at 20 ° C, the reaction vessels were filled with STC medium, mixed and centrifuged at 4 ° C for 10 min. The pellets were resuspended and plated on selective medium osmotically stabilized with sorbitol. The plates were incubated at 30 ° C for 7 days and checked for growth of colonies. The transformation rate from several experiments was 1 - 5 transformants / μg plasmid DNA for Aspergillus niger, 0.1 - 0.5 transformants / μg plasmid DNA for Aspergillus awamori.

Transformants were purified by inoculating on selective plates with acetamide. High copy number transformants were used identified by plating on selective plates with acrylamide. In contrast to acetamide, acrylamide is a poor substrate for the enzyme encoded by the amdS gene and can only support growth in transformants with a high copy number of the amdS gene. Transformants that can functionally express laccase enzyme were identified by first plating on plates with maltodextrin, an inducer for the expression of the glaA promoter, and after two days of growth at 28 ° C. with ABTS agar (0.1% ABTS, 1 % Agarose, in Mclllvaine buffer, pH 4.5). Transformants expressing laccase were indicated by the formation of a green field. Spore suspensions were prepared from transformants which both showed good growth on acrylamide plates and reacted positively with ABTS in the activity test.

8. Example

Expression of the Trametes versicolor Laccase Lac5.5 in

Aspergillus The expression of Laccase Lac5.5 in Aspergillus was in the

Shake flask examined. The following growth medium was used: A 5% (w / v) solution of maltodextrin in tap water was autoclaved for 20 minutes. Then 1 ml 1M IM MgSulfate, 0.5 ml 1000 x trace element solution, 10 ml 50 x Asp A solution and 5 ml 10% (w / v) 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 7 H ₂ 0, 1.1 g of H ₃ B0 ₃ , 0.5 g of MnCl ₂ x 4 H ₂ 0 .0 were dissolved in 80 ml of H ₂ 0. 5 g FeS0 ₄ x 7 H ₂ 0, 0.17 g CoCl ₂ x 5 H ₂ 0, 0.16 g CuS0 ₄ x 5 H ₂ 0, 0.15 g Na ₂ Mo0 ₄ x 2 H ₂ 0 and 5 g EDTA. The 50 × AspA solution had the composition: 150 g of NaNO ₃ , 13 g of KC1, 38 g of KH ₂ PO ₄ , pH 5.5 with 10 M KOH were dissolved in 500 ml of H ₂ O. CuS0 ₄ x 5 H 0 was additionally added to the medium in a final concentration of 0.5 mM.

For expression experiments, 50 ml of the growth medium were inoculated in a 300 ml Erlenmeyer flask with 1 x 10 spores / ml. The cultivation took place in a shaking incubator 30 ° C. and 300 rpm. Samples were taken daily for a week and the laccase activity in the culture supernatant was determined. The maximum laccase activity was observed between 60 and 100 h growth and was between 0.5 and 2.5 U / ml. The laccase activity became colorimetric with the

Substrate ABTS (0.1 mM in the test) was determined in Mclllvaine buffer pH 4.5 and at 37 ° C. Mclllvaine buffer was prepared by titrating a 0.1 M citric acid solution to pH 4.5 with 0.2 M Na ₂ HP0 ₄ solution. The increase in extinction was measured at 420 nm (extinction coefficient of ABTS at 420 nm: 3.6 x 10 ⁴ 1 x Mol x cm). 1 U laccase activity was defined as 1 μmol ABTS substrate turnover / min.

9. Example expression of the Trametes versicolor Laccase Lac5.5 in Pichia pastoris

A commercially available expression system from Invitrogen with the associated expression vectors (pPIC3 and pPIC9) and Pichia pastoris strains (GS115 and KM71) was used. The Pichia pastoris strains GS115 and KM71 are histidine auxotrophic. The expression vectors contained the promoter and terminator of the alcohol oxidase gene A0X1 from Pichia pastoris. The cDNA gene of the Trametes versicolor Laccase Lac5.5 was cloned between these two genetic regulatory elements. The vector pPIC9, located behind the AOX1 promoter, contained a DNA sequence which codes for the signal sequence of the alpha-factor protein from Saccharomyces cerevisiae, followed by a short section of DNA which is used 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. For selection in Pichia pastoris, the vectors contained the HIS4 gene from Pichia pastoris.

A: Construction of a Laccase Lac5.5 expression vector with the signal sequence of Laccase Lac5.5 The plasmid pLac5.5 and the primers K and L were used to amplify the laccase gene. Primers F and G had the following sequence:

Primer K:

5 '-ACTCGAGAATTCACCATGACTGGGCTGCGTCTTCTTCC-3' (SEQ ID NO: 16)

Primer L:

5 '-ACTAGAGCGGCCGCCTATCACTGGTCGTCAGCGTCGAGGGC-3' (SEQ ID NO: 17)

Primer K contained the sequence for an Eco RI interface (underlined) and then the DNA sequence for the first seven amino acids of the Laccase Lac5.5 signal sequence. Primer L contained the sequence for a Not I interface (underlined) and connected to it, in complementary and reverse orientation, the translation stop codon and the DNA sequence for the last 7 amino acids of the Laccase Lac5.5.

PCR amplifications were carried out in accordance with the prior art known to the person skilled in the art. 20 ng pLac5.5 DNA were used in a 50 μl PCR reaction, which also contained 0.5 U vent polymerase, 1 mM MgCl ₂ , 0.2 mM each of the four dNTPs and 100 pmol each of the primers K and L. The other conditions for the specific amplification of the desired PCR product were: 5 min at 94 ° C, followed by 25 cycles of 1 min at 94 ° C, 1 min at 55 ° C and 2 min at 72 ° C. The expected PCR fragment of 1.5 kb size 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 H ₂ 0. 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 ethanol precipitation. The pPIC3 vector prepared in this way was prepared by PCR amplification

DNA fragment of the Laccase Lac5.5 ligated and thus transformed E. coli Top 10F 'cells (Invitrogen). The plasmid DNA was isolated from ampicillin-resistant clones and that cloned 1.5 kb insert identified by restriction digestion with Eco RI and Not I. Of the 12 clones examined, 9 were positive. The vector obtained in this way was designated by 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 the 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 interface (underlined) and then the DNA sequence for the first nine amino acids of the suspected N-terminus of the processed Laccase Lac5.5. The N-terminus of the processed

Laccase Lac5.5 was derived from the sequence comparison with other laccases.

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 the primers L and M. The vector pPIC9 was generated with Eco RI and Not I cut and prepared as the pPIC3 vector described in section A of this example, ligated with the DNA fragment of laccase Lac5.5 produced by PCR amplification and thus transformed E. coli Top 10F 'cells (Invitrogen). 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. Of the 12 clones examined, 3 were positive. The vector thus obtained was named pL532 (Fig. 4).

C: Transformation of Pichia pastoris Pichia pastoris strains GS115 and KM71 were first cultivated in 5 ml of YPD medium (1% yeast extract, 2% peptone, 2% dextrose) at 30 ° C. overnight. With 0.2 ml of this preculture, two main cultures of 250 ml of YPD medium were inoculated and again cultured at 30 ° C. overnight to an optical density (OD600 nm) of 1.3-1.5. The yeast cells of a 250 ml main culture were then centrifuged off (5 min 1500 xg), washed twice with 200 ml H ₂ 0 and once with 10 ml 1 M sorbitol and finally taken up in 0.5 ml 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 at a concentration of 1 μg DNA / μl.

One transformation approach contained 80 μl Pichia pastoris cells and 10 μg linearized vector DNA. Transformation was carried out by electroporation at 1500 V, 25 μF and 200 ohms (BioRad Gene Pulser). The discharge time was approximately 4.2 msec. The transformation mixture was mixed with 1 ml of 1 M sorbitol, incubated for 30 min on ice and then in 0.3 ml aliquots on MD plates without histidine (1.34% YNB, yeast nitrogen base, 4 x 10% biotin, 1% dextrose, 1.5% agar). Transformants appeared after 3-5 days incubation at 30 ° C. Transformants were cleaned by spreading twice on MD plates. Laccase producers were identified on MM indicator plates. MM indicator plates contained 1.34% YNB, 4 x 10 ^"5 % biotin, 0.5% methanol, 1.5% agar, 1 mM ABTS and 0.1 mM Cu sulfate. The inductor methanol was placed in the lid of the petri dishes and Renewed daily to ensure that the colonies were supplied with methanol by diffusion, and laccase producers produced a green yard after 2-3 days of incubation at 30 ° C.

E: Expression in shake flask:

50 ml BMGY medium (1% yeast extract, 2% peptone, 0.1 M K-phosphate, pH 6.0, 1.34% YNB, 4 x 10 ^"5 % biotin, 1% glycerin) were inoculated with a laccase-producing Pichia pastoris transformant and cultured on a shaker at 28 ° C. and 300 rpm for 48 h. The cells of this preculture were isolated by centrifugation (10 min 1500 xg) and suspended in 10 ml 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 further cultivated at room temperature and 300 rpm on a shaker. The main culture was supplemented with methanol (0.3 ml / 10 ml medium) at intervals of 24 h. The production of the recombinant Laccase Lac5.5 started after 24 h. Production rates of up to 4 U / ml were reached after 190 h after the start of the main culture.

10. Example

Isolation of recombinant laccase Lac5.5

Recombinant Laccase Lac5.5 was obtained by growing transformed Aspergillus strains in the shake flask 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. Concentrated Laccase Lac5.5 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.

After cross-flow filtration, concentrated laccase was dialyzed against 20 mM BisTris-HCl, pH 6.5. A conductivity of 1.5 mS / cm was then measured. Dialyzed laccase was then chromatographed on a column of DEAE Sepharose (Pharmacia), equilibrated in 20 mM BisTris-HCl (application buffer), pH 6.5. Under these conditions, the laccase binds to the DEAE Sepharose. When eluting with a linear gradient from 0 to 0.5 M NaCl in application buffer, the laccase activity was found again at a salt concentration of 0.15 M NaCl. The laccase fraction from the DEAE Sepharose column was saturated with ammonium sulfate up to 20% as well mixed with Na acetate, pH 4.5 (final concentration 20 mM) and adjusted to pH 4.5 with acetic acid. The laccase fraction thus prepared was then chromatographed on a column of phenylsepharose (Pharmacia), equilibrated in application buffer (20 mM Na acetate, pH 4.5, 20% saturated ammonium sulfate). Under these conditions, the laccase binds to the phenylsepharose. When eluting with a linear gradient of 20-0% saturated ammonium sulfate, in 20 mM Na acetate, pH 4.5, the laccase activity was found 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 step elution with 0.3 M NaCl. Binding and elution with 0.3 M NaCl were each carried out 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 determined sequence,

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 the mature Laccase Lac5.5 derived from the cDNA sequence and homology comparisons.

Example 11

Biochemical properties of recombinant laccase Lac5.5

The optimum pH and temperature as well as the pH and temperature stability of recombinant Laccase Lac5.5, produced in Aspergillus as described in Example 8, were examined. For these experiments, recombinant Laccase Lac5.5 was first buffered via Sephadex G25 (Pharmacia, PDIO columns) in Mclllvaine buffer, pH 4.5.

A: pH optimum:

For the pH values indicated in FIG. 5, the corresponding buffers were in each case mixed appropriately by unbuffered Na citrate and Na phosphate solutions. For recombinant Laccase Lac5.5, the laccase activity at 37 ° C was then determined at the respective pH. As can be seen from FIG. 5, the Laccase Lac5.5 has an activity optimum in the strongly acidic range for the substrate ABTS.

B: pH stability:

Laccase Lac5.5 was in Mclllvaine buffer at pH 3, 0, 4.5 and

6.0 pre-incubated (temperature 37 ° C). After 0, 30, 60 and 120 min aliquots were taken, diluted in Mclllvaine buffer at pH 4.5 and the laccase activity at 37 ° C determined. At pH 4.5 and 6.0, the laccase activity was not affected by the pretreatment. At pH 3.0, the half-life of the laccase was between 60 and 120 min. (Fig.6)

C: optimum temperature:

The laccase activity of the recombinant Laccase Lac5.5 was determined at the temperatures indicated in FIG. Laccase activity was determined with the ABTS test in Mclllvaine buffer, pH 4.5. It was surprisingly found that the temperature optimum of the Laccase Lac5.5 is 70 ° C. The temperatures at which half-maximum laccase activity was measured are between 50 ° C and 80 ° C.

D: Temperature stability:

Laccase Lac5.5 was pre-incubated in Mclllvaine buffer pH 4.5 at 45, 55 and 65 ° C. After 0, 30, 60 and 120 min aliquots were taken, diluted in Mclllvaine buffer at pH 4.5 and the laccase activity at 37 ° C determined. At 45 ° C the laccase activity was not affected by the pretreatment. At 55 ° C, 80% residual activity was measured after 120 min preincubation. At 65 ° C the half-life of the laccase was 60 min. (Fig.8). SEQUENCE LOG

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Consortium for electrochemical industry GmbH

(B) STREET: Zielstattstrasse 20

(C) LOCATION: Munich

(E) COUNTRY: Germany

(F) POSTCODE: D-81379

(G) TELEPHONE: 089 74844 0 (H) TELEFAX: 089 74844 350

(ii) APPLICATION TITLE: DNA sequences, expression of these DNA sequences by the DNA

Sequences encoded thermophilic laccases and their use

(iii) NUMBER OF SEQUENCES: 19

(iv) COMPUTER READABLE FORM:

(A) DISK: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patentin Release # 1.0, Version # 1.25 (EPA)

(2) INFORMATION ABOUT SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1572 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNS to mRNS

(iii) HYPOTHETICAL: NO

(iii) ANTISENSE: NO (vi) ORIGINAL ORIGIN:

(A) ORGANISM: Trametes versicolor

(B) STEM: TV-1

(vii) DIRECT ORIGIN:

(B) CLON: plac55

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

ATGACTGGGC TGCGTCTTCT TCCTTCCTTC GCGGCGTTGG CCGTGACCGT GTCTCTCGCG 60

CTCAACGCGT TGGCTGGGAT CGGGCCTGTG CTCGACCTCA CGATCTCCAA CGCGGTGGTG 120

TCGCCCGATG GCTTCTCTCG CGCGGCGGTC GTTGCGAACG ACCAGGCTCC CGGGCCTCTC 180

ATTACGGGCC AGATGGGCGA CCGTTTCCAG ATCAATGTGG TCAACAAGCT GTCGAACCAC 240

ACTATGCTCA AGTCGACCAG CATCCACTGG CACGGCTTCT TCCAGAAGGG TACGAACTGG 300

GCCGATGGCC CCGCGTTCGT GAACCAGTGC CCGATCGCGA CTGGTCACTC GTTCCTTTAC

360

GACTTCCAGG TCCCGGACCA GGCCGGGACG TTCTGGTACC ACAGCCATTT GTCTACCCAG

420

TACTGTGACG GGTTGAGAGG TCCTTTCGTC GTCTACGACC CGAACGACCC TCATGCCAGC 480

CTCTACGACG TGGACAACGA TGACACCGTC ATCACCCTCG CTGACTGGTA CCATACCGCT 540

GCCAAGCTTG GGCCGGCCTT CCCTCCTGGC TCTGATGCGA CGTTGATCAA TGGGCTCGGA 600 CGTACAGCGG CCACCCCCAA CGCGGATCTC GCTGTCATTA GTGTCACGCA CGGCAAGCGG 660

TACCGTTTCC GCCTGGTGTC GATGTCCTGC GACCCCGCGT ACACGTTCAG CATCGACGAC 720

CACTCGATGA CCATCATCGA GGCGGACTCA GTCAACACAA AGCCGCTCGA GGTCGACTCG 780

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 AACGTCACCA TTCGCTTCAG GACTGACAAC CCTGGCCCGT GGTTCCTCCA CTGCCACATC 1440

GACTTCCACT TGGAGGCCGG CTTCGCCGTG GTCATGGCTG AAGACATCCC CGACACCAAG 1500

GCCGACAACC CTGTTCCTCA GGCGTGGTCA GACCTTTGCC CCATCTACGA CGCCCTCGAC 1560

GCTGACGACC AG 1572

(2) INFORMATION ABOUT SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS ^: (A) LENGTH: 1572 base pairs (B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNS to mRNS

(iii) HYPOTHETICAL: NO

(iii) ANTISENSE: NO

(vi) ORIGINAL ORIGIN:

(A) ORGANISM: Trametes versicolor

(B) STEM: TV-1

(vii) DIRECT ORIGIN: (B) CLON: plac56

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

ATGACTGGGC TGCGTCTTCT TCCTTCCTTC GCGGCGTTGG CCGTGACCGT GTCGCTCGCG 60 CTCAACGCGT TGGCCGGGAT CGGGCCCGTG CTCGACCTTA CGATCTCCAA TGCGGTTGTT 120

TCGCCCGATG GCTTCTCTCG CGCGGCGGTC GTCGCGAACG 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

GCCAAGCTTG 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 840 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 CTCGTTCCCC GCGACTACTG GTGCTCCCGG TGCCCCCCAC 1260

CCCTTCCACT TGCACGGTCA CACCTTCGCC GTTGTGCGCA GCGCGGGCAG CGCTGAGTAC

1320

AACTACGACA ACCCCATCTG GCGCGACGTC GTCAGCACTG GTACCCCTGC AGCGGGCGAT

1380

AACGTCACCA TTCGCTTCAG GACTGACAAC CCTGGCCCGT GGTTCCTCCA CTGCCACATC 1440

GACTTCCACT TGGAGGCCGG CTTCGCCGTG GTCATGGCTG AAGACATCCC CGACACCAAG 1500

GCCGACAACC CTGTTCCTCA GGCGTGGTCA GACCTTTGCC CCATCTACGA CGCCCTCGAC 1560

GCTGACGACC AG 1572

(2) INFORMATION ON SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 524 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Protein

(iii) HYPOTHETICAL: YES

(vi) ORIGINAL ORIGIN:

(A) ORGANISM: Trametes versicolor

(B) STEM: TV-1

(ix) FEATURES:

(A) NAME / KEY: Protein (B) 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 15

Val Ser Leu Ala Leu Asn Ala Leu Ala Gly Ile Gly Pro Val Leu Asp 20 25 30

Leu Thr Ile Ser Asn Ala Val Val Ser Pro Asp Gly Phe Ser Arg Ala 35 40 45

Ala Val Val Ala Asn Asp Gin Ala Pro Gly Pro Leu Ile Thr Gly Gin 50 55 60

Met Gly Asp Arg Phe Gin Ile Asn Val Val Asn Lys Leu Ser Asn His 65 70 75 80

Thr Met Leu Lys Ser Thr Ser Ile His Trp His Gly Phe Phe Gin Lys 85 90 95

Gly Thr Asn Trp Ala Asp Gly Pro Ala Phe Val Asn Gin Cys Pro Ile 100 105 110 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 Ile 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 Ile Asn Gly Leu Gly Arg Thr Ala Ala Thr Pro Asn Ala 195 200 205

Asp Leu Ala Val Ile 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 Ile Asp Asp 225 230 235 240

His Ser Met Thr Ile Ile Glu Ala Asp Ser Val Asn Thr Lys Pro Leu

245 250 255

Glu Val Asp Ser Ile Gin Ile 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 Ile Asn Ser Ala Ile Leu 290 295 300

Arg Tyr Asp Thr Ala Ser Pro Thr Glu Pro Thr Thr Thr Gin Ala Thr 305 310 315 320 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 Ile Asn 340 345 350

Phe Ala Phe Ser Phe Asn Gly Ser Asn Phe Phe Ile Asn Gly Ala Thr 355 360 365

Phe Gin Pro Pro Thr Thr Pro Val Leu Leu Gin Ile 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 Ile 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 Ile Trp Arg 435 440 445

Asp Val Val Ser Thr Gly Thr Pro Ala Ala Gly Asp Asn Val Thr Ile 450 455 460

Arg Phe Arg Thr Asp Asn Pro Gly Pro Trp Phe Leu His Cys His Ile 465 470 475 480

Asp Phe His Leu Glu Ala Gly Phe Ala Val Val Met Ala Glu Asp Ile 485 490 495

Pro Asp Thr Lys Ala Asp Asn Pro Val Pro Gin Ala Trp Ser Asp Leu 500 505 510

Cys Pro Ile Tyr Asp Ala Leu Asp Ala Asp Asp Gin

515 520

(2) INFORMATION ABOUT SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: YES

(iii) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

TGGCAYGGNT TYTTYCA 17th

(2) INFORMATION ON SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: YES

(iii) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

TCDATRTGRC ARTG 14

(2) INFORMATION ON SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: YES

(iii) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

ATTCAGGGAT CCTGGTAYCA YWSNCAY 27

(2) INFORMATION ABOUT SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: YES

(iii) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

ATACGAGGAT CCRTGNCCRT GNARRTG 27

(2) INFORMATION ABOUT SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3284 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iii) ANTISENSE: NO

(vi) ORIGINAL ORIGIN:

(A) ORGANISM: Trametes versicolor (B) STEM: TV-1

(vii) DIRECT ORIGIN:

(B) CLON: plac56chr

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

TTGGAATGGG CCCGCCGGTG TCGATCGCAA ACAGGTGAGT TCAGTACTAG CCCATCAGCG 60

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 ATCGGGGACG GCGCGTGGCT TGGCACGCGT AGCACCGACG CGCACTATAA AGGCGATGCG 420

GCAGAGAAGA GGCGGAGCAC CACGTTCAGT CCCTTCCTTG GATTCCGGGC AGCTTACTCC 480

TTCTCGCCTC 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 1140 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 AACTTTATGG TTTATAGGTA 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 CCGCGTCGCC GACCGAGCCG ACCACGACGC AGGCCACCTC 1800

TACGAAGCCG TTGAAGGAGA CGGACCTTGA GCCTCTCGCG TCGATGCCGG TGGTAAGTCT 1860

GACTAGCACT TCATCTTTGA TGGTATGCTC ATGCAACTCT CCAGCCTGGC TCTGCTGTGT 1920 CGGGTGGTGT GGACAAGGCG ATTAACTTCG CTTTCAGCTT CGTACGTCCA ACTCACGATT 1980

TCCCCCTTGA GATTTATATT GATGGCTCCA TTGACGGCTC CTCATAGAAC GGGTCCAACT 2040

TCTTCATCAA CGGCGCGACC TTCCAGCCGC CCACCACTCC CGTTCTGCTG CAGATCATGA 2100

GCGGTGCCCA GGCTGCTÄGC 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 AATCGGTCGC TGACAGTCCT

2460

GCAGACTGAC AACCCTGGCC CGTGGTTCCT 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 TCACGAGATC GTCAACCATT TCCTCAATCA TTGACTTACC GACTTGCTAT TTCTAACACG 2760

CTATTTGCGA ACCCCCGCTC TCCCCTCTCT CACACTACGG TCCCTTCGTG AACATGGACT 2820

TGCATGGACT TTGGATTGTA GAAAGTTTAC ACAGCTGTAT AGTCGAATTA TCCCCGTAAT 2880

GCATGGTAGT GCCGCTGGCC TTTACCTCAA TCATTGTTAT CATGATATGG CCATCATAAA

2940

CATCACTGAC ATCTACTAAT CTGCTGTTAG TTTTGGGACC TCAAGAAGAT AAACGCCCGT 3000

CTACCACGAT GTGACGCGCG CGATACGTGA ATGTGACTGA TCGCGTTCCA TTATTCAAAA 3060

CGCGTCGGCT GGCGGCCAGG CCAAGTTGCT CCTCTCTCTC CGACGACGAC CACCCCTGGC 3120

TCTCTTACCC ACCTTCTCTG CACCATGACG GCAGACTACA GACTACAGTC TCTCGACGAT

3180

CCGACGGCGG TCATCCAAGA GCTCTACCGC GCCCATCCAG ACCCGAACGG TTTCCCCCGC

3240

CTCGTTGCTG AGCACTTCCA AAAGCTCTTC GAGAACCGAA CATG 3284

(2) INFORMATION ON SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: YES (iii) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

CCGGAATTCA TGACTGGGCT GCGTCTCCTT CCTTCCTTC 39

(2) INFORMATION ABOUT SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: YES

(iii) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

GAGAGGCCCG GGAGCCTGG 19

(2) INFORMATION ABOUT SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: YES (iii) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

GCTGAATTCG AAGACATCCC CGACACCAAG G 31

(2) INFORMATION ON SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: YES

(iii) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

TGCTCTAGAA AGCTTAAGTT CACTGGTCGT CAGCGTCGAG GG 42

(2) INFORMATION ON SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 43 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: YES (iii) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

CCGGAATTCG ATATCCAAGC GCGGGATCGG GCCTGTGCTC GAC 43

(2) INFORMATION ABOUT SEQ ID NO: 14: 0

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear 5

(ii) MOLECULE TYPE: Peptide

(iii) HYPOTHETICAL: YES

o (v) TYPE OF FRAGMENT: inner

(vi) ORIGINAL ORIGIN: (A) ORGANISM: Aspergillus niger

5

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

Ile Ser Lys Arg 1

° (2) INFORMATION ABOUT SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid 5 (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Peptide (iii) HYPOTHETICAL: YES

(v) TYPE OF FRAGMENT: interior

(vi) ORIGINAL ORIGIN:

(A) ORGANISM: Saccharomyces cerevisiae

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

Glu Lys Arg Glu Ala Glu Ala 1 5

(2) INFORMATION ON SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: YES

(iii) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

ACTCGAGAAT TCACCATGAC TGGGCTGCGT CTTCTTCC 38

(2) INFORMATION ON SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: YES

(iii) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

ACTAGAGCGG CCGCCTATCA CTGGTCGTCA GCGTCGAGGG C 41

(2) INFORMATION ON SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: YES

(iii) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

ACTCGAGAAT TCGGGATCGG GCCTGTGCTC GACCTCACG 39

(2) INFORMATION ABOUT SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Peptide

(iii) HYPOTHETICAL: NO

(v) TYPE OF FRAGMENT: N-terminus

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

Gly Ile Gly Pro Val Leu Asp Leu Thr Ile Ser Arg Ala Val 1 5 10

Claims

claims

1. DNA sequence which codes for a protein with laccase activity, characterized in that it has the DNA sequence SEQ ID N0: 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 one Includes DNA sequence with a sequence homology greater than 80% to the DNA sequences mentioned.

2. Expression vector containing a DNA sequence according to claim 1.

3. Expression vector according to claim 2, characterized in that it additionally contains: a promoter which mediates the expression of the laccase gene in a host organism and suitable signals for the host organism for the transcription termination, the functional with the 3 'end of the DNA Sequence according to claim 1 are linked.

4. Expression vector according to claim 3, characterized in that the promoter is selected from the group tac promoter, subtilisin promoter, GAL promoter, TAKA amylase promoter, polyhedrin promoter, glucoamylase promoter, GAPDH promoter and alcohol oxidase promoter .

5. Expression vector according to claim 3 or 4, characterized in that it additionally contains an N-terminal signal sequence.

6. Expression vector according to claim 5, characterized in that the N-terminal signal sequence is the natural signal sequence contained in the laccase gene or is selected from the group of signal sequences of the following secreted proteins: alpha-cyclodextrin-glucosyltransferase from Klebsieila 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.

7. Expression vector according to claim 5, characterized in that a gene for a secreted protein, or a gene segment for secreted fragment of a protein, is functionally linked to the laccase gene.

8. microorganism strain which contains a DNA sequence according to claim 1 or an expression vector according to any one of claims 2 to 7.

9. Protein comprising the protein sequence SEQ ID NO: 3

10. Use of the protein according to claim 9 for the delignification of pulp, the depolymerization of high molecular aggregates, the deinking of waste paper, the polymerization of aromatic compounds in waste water, especially of lignin-containing waste water from cellulose bleaching, the oxidation of dyes, or the Activation of dyes for pigment formation, use in organic synthesis in coupling reactions of aromatic compounds or the oxidation of aromatic side chains.