CA2292723A1 - Dna sequences, expression of said dna sequences, thermopile laccases coded by said dna sequences and the use thereof - Google Patents

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 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 u:e thereof .
1~0 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 foar 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 suk~strate specificity for aromatic compounds which they oxidize.
Tiie 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 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 pro-karyotic 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 thermophilic properties from the filamentous fungus of th,e subclass of Deuteromycetes, Scytalidium thermo-philum, is described in WO 95/33837. It is not known a whether this enzyme is suitable for pulp bleaching.
As yet, no process for preparing a thermophilic laccase from a filamentous fungus of the subclass of Basidiomycetes as recombinant protein has been described.
CA: AN 96-203142 discloses various characteris-tics of a thermophilic laccase. No DNA or protein sequence of this protein is disclosed.
The invention relates to a DNA sequence which codes for a protein having 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.
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 and 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 reGction 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 5.
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 muta-genesis, 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 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.
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 lactase 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 lactase gene in the best organism. This should preferably be a strong pro:noter~in order to be able to ensure high expression efficiency. The promoter is preferably functionally linked to the 5' end of the lactase gene.
Suitable and preferred promoters are selected from the group of tat 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 tat 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, addition ally 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 rollowing secreted proteins are preferred: alpha-cyclodextrin glucosyl-transferase 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-philic laccase.

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, pyre, trpC, His4, niaD, argB, or hygB.
Particularly suitable as selection markers are amdS and the pyre 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 fi.lamentous 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 ~accases. The pH optimum of the enzyme activity is in the acidic range and is maximal at pH 2.0, with half maximal enzyme activity at pH 4Ø The enzyme stability at 45°C and a pH of 4.5 to 6.0 is i00% for a period of up to 2 h.
The enzyme stability at ~5°C and a pH of 3.0 is 50% for a period of up to 1 h. The temperature optimum at a pH of 4.5 is 70°C. The enzyme activit~.~ at 65 -75°C is still 80% of the maximal activity. The enzyme activity at 50 - 80°C is still 50% of the maximal «ctivity. The enzyme stability at a pH of 4.5 and temperatures up to 55°C is 90% for a period of up t~
2 h. The enzyme stability at a pH of 4.5 and a temperature of 65°C i ~ 50 % for a period of up to 1 h.
The novel proteins comprise the protein sequence SEQ ID NO: 3.
The novel protein is preferably prepared by ehpressing novel DNA sequences in an abovementioned microorganism.
The DNA is preferably expressed using one of the abovementioned expression vectors in the micro-organism.
The invention thus also relates to micro organisms which comprise novel DNA sequences or novel expression vectors.
It is particularly preferable to use com binations 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 - g _ of the thermophilic lactase itself or the glucoamylase portion of the glucoamylase-lactase 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 lactase itself or the signal sequence of the alpha-factor 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 ir~ the detoxification of contaminated soils. Further areas of use relate to the o:~cidation of dyes and the acr_ivation of dyes by reaction with precursor components to form pigments.
Aveas 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 lactase 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-hydroxybenzo-triazole.
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 pigments; the use in organic synthesis for coupling _ g _ 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 rL512.
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 l.accase 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 (3°s malt extract, 0.3%

peptone from soya bean meal, 1.5% agar-agar, pH 5.0) at 28°C for 7 days. Three pieces were punched out of the malt-agar plates and used to inoculate 100 ml of sterile malt extract medium (3% malt extract, 0.3%
peptone from soybean meal, pH 5.0) in 500 ml Erlenmeyer flasks. The culture was incubated at 28°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 '!0 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 ~g of RNA.
600 ~Cg 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 fig. 7.25 ~.g of the isolated mRNA were used to synthesize cDNA. A cDNA synthesis kit from Stratagene was used for Lhis. 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 cJNA 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 105 phages/~cg of vector DNA were obtained from the 0.8 - 2.1 kb fraction. 1 x 105 phages/~Cg of vector DNA ..
were obtained from the 2.1 - 5 kb fraction. The result-ing 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 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 S ml of extraction solution (0.1 M tris-HC1, pH 8.0, 0.1 M EDTA, 0.25 M NaCl, 0.6 mg/ml proteinase K) and 0.5 ml of a l00 (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 incubated at 65°C
for 30 min. After addition of 7 ml of a chloroform/
isoarnyl 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 part by volume of iso-propanol. The precipitated DNA was subsequently puri-~ied on a column (Qiagen Genomic Tip) . It was possible in this way to isolate 0.5 mg of chromosomal DNA f?-orn 16 g of mycelium.
To produce the chromosomal gene bank, 90 ~.g of chromosomal DNA from Trametes versicolor 'rV-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 105 phages/~.g of vector DNA were obtained from the 2 - 4 kb DNA fraction, and 5.4 x 104 phages/ug 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 sequences of known laccase genes: The amino acid sequences of the laccase genes, contained in the EMBL

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 com pared. It was possible through the comparison of sequences to identify four peptides with a length of 5 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: 5) 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 Sam HI cleavage site (underlined) and, attached therzto, in each case the appropriate degenerate lac:case 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 ~1 PCR reaction which additionally contained 1.25 U
cf Taq polymerase, 1.25 mM MgCl2, 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 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 ~.1 from the first PCR reaction was employed in a second PCR reaction which additionally contained 1.25 U of Taq polymerase, 1.25 mM MgCl2, 0.2 mM of each of the four dNTPs and, in each case, 100 pmol of primers C and D. The further conditions for the specific amplication of the required 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. A PCR product of about 1.1 kb was obtained. The PCR productwwas purified by agarose gel electrophoresis, cut with the restriction enzyme Bam HI-, and cloned into a pUCl8 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 electro phoresis and radiolabeled with a- [3zP] -dATP (random priming kit, Boehringer Mar_nheim). Free radioactivity was removed by chromaLOgraphy on Sephadex G25 (Pharmacia). The specific activity of the radiolabeled DNA probe was 1 x '10' cpm/~g .,o.f 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°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 45°C in a hybridization buffer containing 50% formamide.
Positive clones were picked and purified by repetition 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 lactase 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 lactase gene identical in amino acid sequence. The two lactase cDNA genes were called Lac5.5 and Lac5.6. Correspondingly, the plasmids with the two lactase cDNA genes were called pLac5.5 and pLac5.6.
5th example Isolation of the chromosomal gene of a lactase from Trametes versicolor TV-'1 Screening for chromosomal lactase 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 lactase-specific probe described in the 3rd example was used. The hybridization temperature was 45°C in a hybridization buffer containing 50%
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 lactase Lac5.6. The coding region of a clone and, in each case, about 1 kb of flanking sequence in the 5' and 3' regions were sequenced. (SEQ ID NO: 8).

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 she glucoamylase gene (g:laA) from Aspergillus niger (J. C. 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), 8-~ - 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 (M. P. 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 (E. J. 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 Lac5.5 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 pUCl9. To this end, the Lac5.5 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 pUCl9 vector which had previously been cut with Eco RI and Xba I.
The resulting 4.6 kb plasmid was called pLacS.

To modify the start ATG codon of the Lac5.5 cDNA gene, primers E and F were used.
Primer E: 5'-CCGGAATTCATGACTGGGCTGCGTCTCCTTCCTTCCTTC-3' S (SEQ ID NO: 9) Primer F: 5'-GAGAGGCCCGGGAGCCTGG-3' (SEQ ID NO: 10) Underlining in primer E indicates a Bsp HI
clea~rage 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
N0: 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 by 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 1.10 by in size in the 3' region of the Lac5.5 cDNA gene. A 100 ~l PCR mixture contained in each case 10 ng of Lac5.5 cDNA (in the pBK CMV vector), 0.5 U of Tth polymerase, 1.25 mM MgClz, 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 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 primers E and F was first cut with Eco RI and Xma I and then purified by gel electrophoresis and cloned into the vector pLacS which had previously been cut with Eco RI and Xma I. This resulted in the vector pLac51 in 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 pUCl9 vector which had previously been cut with Eco RI
and Xba I. The insert about 100 by in size was cut out of the resulting plasmid pLTS 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 lactase cDNA gene.
The cDNA gene for the Trametes versicolor lactase 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 )cb fragment which contained the coding region of the Lac5.5 cDNA gene and about 1.1 kb of the pUCl9 vector. This fragment was cut in a second step with Afl II, and r_he resulting Lac5.5 cDNA fragment 1.5 kb in size was isolated. This f ragment was 1 igated into the Af 1 I I - Nco T fragment 'i.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 pUCl8 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 lactase cDNA gene B: Linkage of the lactase Lac5.5 cDNA to the crlaA
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 lactase Lac5.5, primers I
and F were used.

Primer I: 5'-CCGGAATTCGATATCCAAGCGCGGGATCGGGCCTGTGCT-CGAC-3' (SEQ ID N0:13) S 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 by 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 by in size 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 cut with Eco RI and Xma I and then purified by gel electrophoresis and cloned :intc the vector pLacS 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 S' region in front of the codon for the first amino acid of the mature lactase protein, an Eco RV cleavage site followed by the codons for the amino acids of the sequence Ile Ser Lys Arg (SEQ ID N0:14) . This sequence is a recognition site for KEX2 protease. The 3' region of the lactase cDNA gene in the vector pLac52 was modified as described for the vector pLac5l. The insert about 100 by 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 lactase cDNA gene.
The cDNA gene for the Trametes versicolor lactase 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

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 pUCl8 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 glucoarnylase including signal sequence, of the recognitior_ sequence for the :r~EX2 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 pyre selection markers ~.nto the vectors pANlacl and pANlac2 pANlacl and pANlac2 were linear~ized by digestion of 5 ~g 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 pyre (orotidine-5'-monophosphate 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 ~g of each of the linearized and phosphatase-treated vectors pANlacl and pANlac2 were ligated to 0.6 ~g of the isolated Not I fragment, and 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. 2). 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 (pyre-) (W. van Harti.ngsveldt, I.E. Mattern, C.M.J. van Zeijl, P.H. Pouwels and C.A.M.J.J. van den Hondel (1987) D~Iol.
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 (P. 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°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 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-HC1, pH 7.5). Protoplasts were isolated by 5 centrifugation of the suspension in a sterile sample vessel (2000 rpm, 4°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 10 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 pyre 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 ~g of plasmid DhTA in incubation vessels with a volume of 12 ml and incubated on ice for 25 min. Then 2G 1.25 ml of a 60o PEG4000 solution (60°s PEG4000, 50 mM
calcium chloride, 10 mM tris-HC1, pH 7.5) were slowly added with repeated mixing to the transformation mixture. After incubation at 20°C for a further 20 min, the reaction vessels were filled with STC medium, mixed and centrifuged at 4°C for 10 min. The pellets were resuspended and plated out 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 on several experiments was 1 - 5 transformants/ug of plasmid DNA
for Aspergillus niger, and 0.1 - 0.5 transformants/~.g 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 copy number of the amdS gene. Transformants capable of functional expression of the lactase 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°C for 2 days, covered with ABTS agar (0.1% ABTS, 1% agarose, in McIllvaine buffer, pH 4.5). Lactase-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 lactase Lac5.5 in Aspergillus Expression of lactase Lac5.5 in Aspergillus was investigated on the shaken flask scale. The following culture medium was used: a 5% (w/v) 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% (w/v) casamino acids were added to 50U ml of this basic medium. The 1000 x trace element solution had the following composition: 2.2 g of ZnS04 x 7Hz0, 1.1 g of H3B03, 0.5 g of MnCl2 x 4H20, 0.5 g of FeS04 x 7H20, 0.17 g of CoClz x 5H20, 0.16 g of CuS04 x 5H20, 0.15 g of Na2Mo04 x 2Hz0 and 5 g of EDTA were dissolved in 80 ml of H20. The 50 x AspA solution had the com-position: 150 g of NaN03, 13 g of KC1, 38 g Of KH2PO4 were dissolved in 500 ml of HZO, pH 5.5 adjusted with 10 M KOH. CuS04 x 5Hz0 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°C and 300 rpm.
Samples were taken each day for one week, and the lactase activity in the culture supernatant was deter-mined. The maximum lactase activity was observed with growth between 60 and 100 h, and was between 0.5 and 2.5 U/ml. The laccase activity was determined by colorimetry with the substrate ARTS (0.1 mM in the assay) in McIllvaine buffer pH 4.5 and at 37°C.
McIllvaine buffer was prepared by titrating a 0.1 M
citric acid solution against 0.2 M Na2HP04 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-1 x cm~l) . 1 U of laccase activity was defined as 1 ~cmol 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 (pFIC3 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 exidase 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 3C ampicillin-resistance gene for selection in E. coli.
The vectors contained the HI54 gene from Pichia pastoris for selection in Pichia pastoris.
A: Construction of a laccase Lac5.5 expression vector with the signal sequence of laccase Lac5.5 The plasmid pLac5.5 and primers K and L were used for amplification of the laccase gene. Primers F
and G had the following sequence:

Primer K:
S'-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 Lac5.5 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 Lac5.5.
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 ~C1 PCR reaction which additionally contained 0.5 U of Vent polymerase, 1 mM MgClz, 0.2 mM of each of the four c.'NTPs and in each case 100 pmol of primers K and L. The ether conditions for the specific amplification of the required PCR product were: 5 min at 94°C foJ.lowed by >5 cycles of 1 min at 94°C, 1 min at 55°C and 2 min at 72°C. Th2 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 phosphatas2. 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 lOF' 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. 9 of 12 investigated clones were positive. The vector obtained in this way ' was given the name pL512 (Fig. 3).
B: Construction of a laccase Lac5.5 ex ression vector ;with the signal sequence of the al ha-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 N
terminus 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 lOF' 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 (1% yeast extract, 2% peptone, 2% dextrose) at 30°C overnight.
0.2 ml portions of this preculture were used to inoculate two main cultures each of 250 ml of YPD
medium, and cultivation was again carried out at 30°C
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 H20 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, precipi-tated with ethanol and taken up in H20 in a concen-tration of 1 ug of DNA per ~cl. A transformation mixture contained 80 ~cl of Pichia pastoris cells and 10 ~.g of linearized vector DNA. Transformation took place by electroporation at 1500 V, 25 ~CF and 200 Ohm (BioRad Gene Pulser). The discharge time was about 4.2 msec.
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-~%
:~iotin, 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. Lactase 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 rnM 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. Lactase producers generated a green halo after incubation at 30°C for 2 - 3 days.
E: Expression in shaken flasks:
50 ml of BMGY medium (1% yeast extract, 2%
peptone, 0.1 M K phosphate, pH 6.0, 1.34% YNB, 4 x 10-S%
biotin, 1% glycerol) were inoculated with a laccase producing Pichia pastoris transformant and cultivated on a shaker at 300 rpm and 28°C for 48 h. The cells from this preculture were isolated by centrifugation (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-S% 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 lactase Lac5.5 started after 24 h. Production rates of up to 4 U/ml were reached 190 h after starting the main culture.
10th example Isolation of recombinant lactase Lac5.5 Recombinant lactase Lac5.5 was obtained by :15 culturing transformed Aspergillus strains in shaken flasks as described in the 8th example. Culture supernatants containing lactase Lac5.5 were concen trated by cross-flow filtration. Sartocon micro ziltration modules (Sartorius) with an exclusion' limit of 30 kD were used for this. Concentrated lactase hac5.5 was then lyophilized and dissolved in 20 mM Na phosphate, pH 6Ø The activity of the concentrated r'combinant lactase Lac5.5 was 18.6 U/ml.
Lactase 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 lactase was then chromatographed on a column of DEAE-Sepharose (Pharmacia), equilibrated in 20 mM
bistris-HC1 (loading buffer), pH 6.5. Under these conditions, the lactase binds to the DEAF-Sepharose. On elution with a linear gradient from 0 to 0.5 M NaCl in loading buffer, the lactase activity was recovered at a salt concentration of 0.15 M NaCl. The lactase fraction from the DEAF-Sepharose column was mixed with ammonium sulfate to 20% saturation and with Na acetate, pH 4.5 (final concentration 20 mM), and the pH was adjusted to 4.5 with acetic acid. The lactase fraction prepared in this way was then chromatographed on a column of phenyl-Sepharose (Pharmacia) equilibrated in loading 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 DEAF-Sepharose and subsequent stepped elution with 0.3 M NaCl. Binding and elution with 0.3 M NaCl each took place in 20 mM
bistris-HC1, 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 (SE9 ID NO: 19) , Tvas in agreement with the N terminus of mature ~accase Lac5.5 derived From the cDNA sequence and homology comparisons.
11th example Biochemical properties of recombinant laccase Lac5.5 The pH and temperature optimum and the pH and temperature stability of recombinant laccase Lac5.5, 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, PD10 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 Lac5.5 was then determined at each pH at 37°C. As is evident from Fig. 5, the laccase Lac5.5 has an activity optimum in the strongly acidic range for the substrate ABTS.
B: pH stability:
Lactase Lac5.5 was preincubated in McIllvaine buffer at pH 3.0, 4.5 and 6.0 (temperature 37°C).
Aliquots were taken after 0, 30, 60 and 120 min and diluted in McIllvaine buffer, pH 4.5, and the lactase activity was determined at 37°C. The lactase activity was not adversely affected by the pretreatment at pH 4.5 and 6Ø The half-life of the lacrase at pH 3.0 was between 60 and 120 min (Fig. 6).
C: Temperature optimum:
The lactase activity of the recombinant lactase Lac5.5 was determined at the temperatures indicated in Fig. 7. The lactase activity was determined using the ABTS assay in Melllvaine buffer, pH 4.5. Surprisingly, i'c was .found from this that the temperature optimum of the lactase Lac5.5 is 70°C. The temperatures at which the measured lactase activity is still half the rnaximum were 50°C and 80°C.
D: Temperature stability:
Lactase Lac5.5 was preincubated in McIllvaine buffer, pH 4.5, at 45, 55 and 65°C. Aliquots were taken after 0, 30, 60 and 120 min and diluted in McIllvaine ..
buffer, pH 4.5, and the lactase activity was determined at 37°C. The lactase activity was not adversely affected by the pretreatment at 45°C. Measurement after preincubation at 55°C for 120 min showed 80o activity still remaining. The lactase half-life at 65°C was 60 min (Fig. 8) .

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Consortium fuer elektrochemische Industrie C~nbH
(B) STREET: Zielstattstrasse 20 (C) CITY: Munich (D) COUNTRY: Germany (E) POSTAL CODE: D-81379 (ii) TITLE OF INVENTION: DNA sequences, expression of these DNA
sequences, thermophilic laccascs encoded by the DNA sequences, and the use thereof (iii) NUMBER OF SEQUENCES: 19 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: McFadden Fincham (B) STREET: 606-225 Metcalfe Street (C) CITY: Ottawa (D) STATE: Ontario (E) COUNTRY: Canada (F) ZIP: K2P 1P9 (v) COMPUTER READABLE FORM:
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(B) FILING DATE: June 4, 1998 (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/EP98/03343 (B) FILING DATE: June 4, 1998 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: McFadden Fincham (B) REGISTRATION NUMBER: 3083 (C) REFERENCE/DOCKET NUMBER: 1546-317 (x) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (613) 234-1907 (B) TELEFAX: (613) 234-5233 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1572 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA
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(A) ORGANISM: Trametes versicolor (B) STRAIN: TV-1 (vii) IMMEDIATE SOURCE:
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GACGGACCTT

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TGCGTCTTCT
TCCTTCCTTC

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(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 Val Ser Leu Ala Leu Asn Ala Leu Ala Gly Ile Gly Pro Val Leu Asp Leu Thr Ile Ser Asn Ala Val Val Ser Pro Asp Gly Phe Ser Arg Ala Ala Val Val Ala Asn Asp Gln Ala Pro Gly Pro Leu Ile Thr Gly Gln Met Gly Asp Arg Phe Gln Ile Asn Val Val Asn Lys Leu Ser Asn His Thr Met Leu Lys Ser Thr Ser Ile His Trp His Gly Phe Phe Gln Lys Gly Thr Asn Trp Ala Asp Gly Pro Ala Phe Val Asn Gln Cys Pro Ile Ala Thr Gly His Ser Phe Leu Tyr Asp Phe Gln Val Pro Asp Gln Ala Gly Thr Phe Trp Tyr His Ser His Leu Ser Thr Gln Tyr Cys Asp Gly Leu Arg Gly Pro Phe Val Val Tyr Asp Pro Asn Asp Pro His Ala 5er Leu Tyr Asp Val Asp Asn Asp Asp Thr Val Ile Thr Leu Ala Asp Trp Tyr His Thr Ala Ala Lys Leu Gly Pro Ala Phe Pro Pro Gly Ser Asp lao 185 190 Ala Thr Leu Ile Asn Gly Leu Gly Arg Thr Ala Ala Thr Pro Asn Ala Asp Leu Ala Val Ile Ser Val Thr His Gly Lys Arg Tyr Arg Phe Arg Leu Val Ser Met Ser Cys Asp Pro Ala Tyr Thr Phe Ser Ile Asp Asp His Ser Met Thr Ile Ile Glu Ala Asp Ser Val Asn Thr Lys Pro Leu Glu Val Asp Ser Ile Gln Ile Phe Ala Gly Gln Arg Tyr Ser Phe Val Leu Glu Ala Asn Gln Asp Val Gly Asn Tyr Trp Val Arg Ala Asp Pro Leu Phe Gly Thr Thr Gly Phe Asp Gly Gly Ile Asn Ser Ala Ile Leu Arg Tyr Asp Thr Ala Ser Pro Thr Glu Pro Thr Thr Thr Gln Ala Thr Ser Thr Lys Pro Leu Lys Glu Thr Asp Leu Glu Pro Leu Ala Ser Met Pro Val Pro Gly Ser Ala Val Ser Gly Gly Val Asp Lys Ala Ile Asn Phe PheSer Phe GlySer Phe Phe Ile Gly Thr Ala Asn Asn Asn Ala Phe ProPro Thr ProVal Leu Gln Ile Ser Ala Gln Thr Leu Met Gly Gln AlaSer Asp LeuPro Gly Asp Val Ala Pro Ala Leu Ser Tyr Leu Ser SerThr Ile LeuSer Pro Ala Thr Gly Pro Asp Glu Phe Thr Ala Gly ProHis Pro HisLeu Gly His Thr Ala Val Ala Phe His Phe Val Arg AlaGly Ser GluTyr Tyr Asp Asn Ile Arg Ser Ala Asn Pro Trp Asp ValSer Thr ThrPro Ala Gly Asp Val Ile Val Gly Ala Asn Thr Arg ArgThr Asp ProGly Trp Phe Leu Cya Ile Phe Asn Pro His Hia Asp HisLeu Glu GlyPhe Val Val Met Glu Ile Phe Ala Ala Ala Asp Pro ThrLys Ala AsnPro Pro Gln Ala Ser Leu Asp Asp Val Trp Asp Cys IleTyr Asp LeuAsp Asp Asp Gln Pro Ala Ala (2) INFORMATION FOR SEQ ID NO: 4:
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(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 Base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: YES
(iii) [sic] ANTISENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

(2) INFORMATTON FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 Base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: YES
(iii) [sic] ANTISENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 Base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNS
(iii) HYPOTHETICAL: YES
(iii) [sic] ANTISENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 Base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: YES
(iii) (sic] ANTISENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: Amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Peptide (iii) HYPOTHETICAL: YES
(v) FRAGMENT TYPE: Internal (vi) ORIGINAL SOURCE:
(A) ORGANISM: Aspergillus niger (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Ile Ser Lys Arg (2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids (B) TYPE: Amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Peptide (iii) HYPOTHETICAL: YES
(v) FRAG~NIENT TYPE: Internal (vi) ORIGINAL SOURCE:
(A) ORGANISM: Saccharomyces cerevisiae (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Glu Lys Arg Glu Ala Glu Ala (2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 Base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: YES
(iii) [sic] ANTISENSE: NO

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

(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 Base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: YES
(iii) [sic] ANTISENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 Base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (E) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: YES
(iii) [sic] ANTISENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

(2) INFORMATION FOR 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) FRAGMENT TYPE: N-Terminus (xi~ SEQUENCE DESCRIPTION: SEQ ID NO: 19:
Gly Ile Gly Pro Val Leu Asp Leu Thr Ile Ser Arg Ala Val

Claims (10)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A DNA sequence which codes for a protein having 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.

5. 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 5, wherein the N-terminal signal sequence is the natural signal sequence present in the laccase gene, or is 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.

7. An expression vector as claimed in claim 5, 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. A protein comprising the protein sequence SEQ ID NO: 3.

10. 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.