KR20010013482A - Dna sequences, expression of said dna sequences, thermopile laccases coded by said dna sequences and the use thereof - Google Patents

Abstract

The present invention consists of a DNA sequence (SEQ ID NO: 1, SEQ ID NO: 2), and relates to a DNA sequence coding for a protein having laccase activity.

The present invention also relates to the expression of DNA sequences, thermophilic laccases and lignin encoded by the DNA sequences, depolymerization of polymer aggregates, depolymerization of waste paper, waste inks, particularly in waste fluids resulting from cellulose bleaching. It relates to the polymerization of aromatic compounds, their use in the production of pigments by oxidizing and activating colorants and their use in organic synthesis used in coupling reactions of organic compounds or side chain oxidation of aromatic compounds.

Description

DNA sequence, expression of the DNA sequence, thermophilic laccase encoded by the DNA sequence, and use thereof {DN SEQUENCES, EXPRESSION OF SAID DNA SEQUENCES, THERMOPILE LACCASES CODED BY SAID DNA SEQUENCES AND THE USE THEREOF}

One of the enzymes of great interest for industrial use is laccase enzyme (p-hydroxy phenol oxidase, EC 1.10.3.2).

Lacase is a family of genes called "blue copper proteins," or families, and is located in three copper centers, generally designated as type 1 to type 3. Contains four copper ions.

Laccases are typically characterized by secreting proteins, with 10-45% of their molecular weight having glycosylation content.

Lacase has a wide range of substrate specificities for oxidizing aromatics.

The electrons from this oxidation reaction are used to reduce oxygen, resulting in water.

The action of laccases in white rot fungi destroys lignin.

It also attracted great interest in the use of laccases in papermaking for the deligination of pulp.

In addition to depolymerization of high molecular compounds such as lignin, laccases can also accelerate polymerization of aromatic compounds.

An example of this is the biosynthesis of lignin in plants containing laccases occurring in plants.

Thus, the industrially possible use of laccases is generally known in various types of polymerization reactions, eg wastewater treatment.

The use of laccases in organic chemical synthesis is known, for example, in coupling or branched chain oxidation of aromatic compounds.

However, in most of their likely uses most known laccases have the drawback of being mesophilic.

That is, these laccases are suitable for low temperature and are limited in thermal stability.

The prerequisite for the industrial use of laccase enzymes is that they can be produced at low cost.

This is generally possible only by the use of enzymes produced by recombinant techniques.

Various prokaryotic and eukaryotic expression systems are available for the production of proteins.

Examples of prokaryotic expression systems are Escherichia coli and Bacillus substilis.

Eukaryotic expression systems that are widely used include cell culture systems of mammalian cells and insect cells and yeast or filamentous eukaryotic microorganisms.

Patent document WO96 / 00290 describes five laccase genes obtained from the subclass of Baidiomycetes, polyporus pinstitus.

One of these laccase genes (LCCI) was made with recombinant proteins.

The thermophilic properties of this enzyme were no longer investigated, but the use of LCC1 laccases for hair dyeing suggested that the enzyme had mesophilic properties.

Patent document WO95 / 33837 describes the preparation of recombinant laccases with thermophilic properties obtained from subclasses of Deuteromycetes, filamentous fungi, scytalidium thermophilum.

It is not known whether this enzyme is suitable for pulp bleaching.

To date, no method for producing thermophilic laccase from subtilis of Basidiomycetes as a recombinant protein has been described in various documents.

Reference CA: AN 96-203142 describes various properties of thermophilic laccases.

The DNA or protein sequence of this protein has been published.

The present invention relates to DNA sequences encoded for proteins having laccase activity, expression of these DNA sequences, thermophilic laccases encoded by the DNA sequences, and the use thereof.

1 shows the structure of the DNA vector pANlac1S.

2 shows the structure of the DNA vector pANlac2S.

3 shows the structure of a DNA vector pL512.

4 shows the structure of a DNA vector pL532.

5 shows the activity dependency of new laccases on pH.

Figure 6 shows the pH stability of the new laccase.

7 shows the activity dependence of new laccases on temperature.

8 shows the temperature stability of the new laccase.

The present invention is explained in more detail with reference to the following examples.

Standard methods and bacterial transformation, Southern and other methods used in DNA or RNA treated examples, such as treatment with restriction endonucleases, DNA polymerases, reverse transcriptases, and the like. Standard methods such as Northern analysis, DNA sequencing, radiolabeling, screening and PCR techniques were performed according to the manufacturer's recommendations of the used kit unless otherwise indicated. If not, it was carried out according to the conventional art known from standard textbooks.

Example 1:

Preparation of cDNA Bank on Trametis Versicolor TV-1

Strain tramethis versicolor TV-1 was used.

First, mycelium was obtained from a Tramethis versicoller on a malt-agar plate (3% malt extract, 0.3% peptone obtained from soy flour, 1.5% agar, pH5.0) at 28 ° C. for 7 days.

Three pieces were punched out of the malt-agar plate, and 100 ml of sterile malt extract medium (3% malt extract, 0.3% peptone obtained from soy flour, pH 5.0) was incubated in a 500 ml Erenmeyer flask.

The culture was incubated at 28 ° C. with stirring at 100 rpm for 7 days.

In this way, the prepared micellium suspension was filtered through a porcelain funnel, washed with 0.9% saline solution, and the microlium was frozen in liquid nitrogen and pulverized with pestle and mortar.

RNA was isolated using RNeasy Kit (Qiagen).

Yield at 200 mg of micelles was 100 μg of RNA.

MRNA was isolated using 600 μg RNA.

This was done by chromatography on oligo-dT Sepharose (mRNA separation kit (Kit, pharmacia)).

The yield of mRNA was 26 μg.

CDNA was synthesized using 7.25 μg of isolated mRNA.

Stratagene cDNA synthesis kit (Kit) was used for this synthesis.

After fractionation, the cDNA was fractionated into a size range of 0.8-2.1 kb and 2.1-5 kb by agarose gel electrophoresis.

Two fractions of cDNA were isolated from the agarose (Qiagen gel extraction Kit) to prepare a cDNA bank.

The cDNA banks were prepared in lambda pharges (Stratagene, Zap Express Cloning System).

Vector DNA 4 × 10 ⁵ phages / μg were obtained at 0.8-2.1 kb fractions.

Vector DNA 1 × 10 ⁵ phage / μg was obtained in a 2.1-5 kb fraction.

The resulting phage was amplified by infecting the E. coli strain XL-1 Blue MRF '(Stratagene).

Example 2

Preparation of Chromosome Gene Bank in Trametis Versicolor

Mycelium was prepared in Tramethis versicolor TV-1 as in Example 1.

The micellium was filtered by suction through a porcelain funnel and washed with 0.9% saline.

Then, frozen in liquid nitrogen, crushed with mortar and pestle, divided into 1g each, 1g of the crushed micelles were taken in a sterile sample container and immediately extracted with a solution (0.1M Tris-Hcl, pH8.0, 0.1M EDTA). , 0.25 M NaCl, 0.6 mg / ml proteinase K) 5 ml and 10% (w / v) sodium lauroyl sarcosine solution 0.5 ml.

After incubation at 50 ° C. for at least 2 hours, the mixture was mixed with 0.85 mL of 5 MNaCl and 7 mL of a solution of 10% (w / v) CTAB solution in 0.7 M Nacl and incubated at 65 ° C. for 30 minutes.

7 ml of a chloroform / isoamyl alcohol mixture (24: 1) was added, and the obtained mixture was stirred to separate the two phases 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 columns (Qiagen Genomic Tip).

In this way, 0.5 mg of chromosomal DNA could be separated from 16 g of micelles.

In order to prepare a chromosome gene bank, 90 µg of chromosomal DNA was completely cleaved with ECORI in tramethisversicollar TV-1 and fractionated by agarose gel electrophoresis.

The chromosomal DNA fractions were separated in the range of 2-4 kb and 4 kb-10 kb and cloned into lambda phage (stratagene, ZAP Express cloning system) in each case.

Vector DNA 1 × 10 ⁵ phage / μg was obtained at 2-4 kb DNA fraction, and vector DNA 5.4 × 10 ⁴ phage / μg was obtained at 4-10kb DNA fraction.

The phage was amplified by infecting the E. coli strain XL-1 Blue MRF '.

Example 3

Preparation of laccase-specific DNA probes from genomic DNA of Trametis versicollar

DNA probes separating the laccase enzymes were prepared by PCR amplification with degenerate primers in the Tramethis versicoller genomic DNA.

The degenerating primers were constructed based on sequence comparison of known laccase genes.

Specifics in the subclass of Neurospora crassa, Coriolus hirsutus, Plelebia radiata, Agaricus bisporus and Basidiomycetes. The amino acid sequence of laccase gene in EMBL gene databank was compared in filamentous fungi.

Through the sequence comparison, it was possible to identify four peptides having a length of 5 to 7 amino acids completely preserved in all laccases.

These peptides were translated into DNA, taking into account codons to produce degenerate primers.

The parameter has the following arrangement:

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 included a Bam HF cleavage site (underlined) at the 5 'end, in which case a suitable degenerate laccase array was attached to that site.

Genomic DNA obtained from Tramethis versicolor was isolated from micelles of the stirred flask culture as in Example 2.

PCR amplification was carried out by conventional techniques well known to those skilled in the art.

In the first PCR reaction, 200ng of chromosomal tramethicoseciccor DNA in 100μl PCR reaction containing Taq polymerase 1.25U, 1.25mM MgCl ₂ , 4 dNTPs each 0.2mM and in each case 100 pmol of primers A and B Was used.

Other conditions of the desired amplification of the PCR product required are as follows:

After 5 minutes at 94 ° C., 0.5 cycles at 94 ° C., 1 minute at 40 ° C. and 2.5 minutes at 60 ° C. and 0.5 cycles at 94 ° C., 0.5 minutes at 94 ° C., 1 minute at 50 ° C. and 30 cycles of 2.5 minutes at 72 ° C. .

1 μl of the first PCR reaction was used in the secondary PCR reaction which further included Taq polymerase 1.25U, 1.25 mM MgCl ₂ , each of 4 dNTPs 0.2 mM and in each case 100 pmol of primers C and D.

Another condition for the desired amplification of the required PCR product is as follows:

5 minutes at 94 ° C., then 0.5 minutes at 94 ° C., 1 minute at 40 ° C. and 2.5 minutes at 60 ° C., 0.5 minutes at 94 ° C., 1 minute at 50 ° C., and 30 minutes at 2.5 ° C. for 30 minutes box.

About 1.1 kb of PCR product was obtained.

The PCR product was purified by agarose gel electrophoresis, digested with restriction enzyme Bam HI, cloned in pUC18 vector digested with restriction enzyme Bam HI, and transformed in E. coli.

The plasmid was isolated from the culture of transformed E. coli.

DNA sequence analysis at the 5 'and 3' ends confirmed that the cloned DNA fragment was a fragment of the laccase gene.

To prepare the DNA probes for screening laccase genes, the laccase-specific PCR fragments were digested with Bam HI, cleaved by agarose electrophoresis and separated to α- [ ³² P. ] -dATP (random priming kit, Boehringer Mannheim, Germany) was radiolabeled.

Free radioactivity was removed by chromatography on Sephadex G25 (Pharmacia).

The specific radioactivity of the radiolabeled DNA probe was 1 × 10 ⁷ cpm / μg of DNA.

Example 4

Isolation of Lacase cDNA Gene from Trametis Versicoller TV-1

The cDNA gene bank was used in the Trametis Versicoller TV-1 described in Example 1.

Screening of laccase cDNA genes was carried out by the prior art.

In the primary screening, the E. coli XL-1 Blue MRF 'cells were first cultured on 10 Petri dishes and then infected with 50,000 cDNA banks per petri dish (0.8-2.1 kb fraction, see Example 1). ).

After overnight incubation at 37 ° C., freshly generated phages were transferred to a nylon filter (Stratagene).

The filter was then hybridized with laccase-specific probes radiolabeled according to the manufacturer's instructions (see Example 3).

The hybridization temperature was 45 ° C. in the hybridization buffer solution containing 50% formamide.

Positive clones were taken and the screening process repeated and purified.

After three separations, 20 strongly hybridized phageclones were isolated in this screening and recloned in the pBK CMV vector by in vivo excision according to the manufacturer's protocol (Straagene).

Analysis of clones and DNA sequences with restriction endonucleases by digestion indicated that nearly two laccasease genes were isolated at that DNA level.

The complete DNA sequence of the two clones indicated that their clones were alleles of the same laccase gene in the amino acid sequence.

These two laccase cDNA genes are shown as Lac5.5 and Lac5.6, and correspondingly the plasmids with these two laccase cDNA genes are shown as pLac5.5 and pLac5.6.

Example 5

Chromosome Gene Isolation of Lacase from Tramethis Versicollar TV-1

As described in Example 2, screening for the cDNA clone described in Example 4 was screened for the chromosomal laccase gene of the chromosomal gene bank in Tramethis vercyclo TV-1 (2-10 kb fraction, see Example 2). The same procedure was followed.

The radiolabeled laccase-specific probe described in Example 3 was used again.

The hybridization temperature was 45 ° C in a 50% formamide-containing hybridization buffer liquid.

In this screening, hybridized phage clones were isolated at three drops and re-cloned in the pBK CMV vector (Stratagene) by in vivo cleavage according to the manufacturer's protocol (stratagene).

Analysis by restriction nucleic acid intermediate enzyme showed that all three clones were identical and had a length of about 7 kb.

Analysis by DNA sequence indicated that all three clones corresponded to the chromosomal gene of laccase Lac5.6.

About 1 kb of the contiguous array was arranged in the coding region of the clone and in each case the 5 'and 8' regions (SEQ ID NO: 8).

Example 6

Preparation of DNA Structure for Expression of Lacase Lac5.5 in Aspergillus

The cDNA of Trametis versicolor laccase Lac5.5 was functionally linked to expression signals specific for filamentous fungi in the Aspergillus family.

The following gene expression components were used in Aspergillus:

a) Promoters of the glucoamylase gene (glaA) in 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), 84-92).

b) DNA fragments and mature glucoamylase fragments encoding the signal sequence following the use of a glaA promoter.

The DNA sequence encoding the KEX2 protease cleavage site is linked to it.

(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) Transcription termination code of trpC gene in Aspergillus nidurans (E.J.

Mullaney, J.E. Hamer, M.M. Yelton and W.E. Timberlake (1985), Mol. Gen.

Genet. 199, 37-45).

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

A: Association of laccase Lac5.5 cDNA to glaA promoter:

Lac5.5 cDNA gene was cloned back into vector pUC19 for further processing.

For this purpose, the Lac5.5 cDNA gene was isolated from the pBKCMV vector obtained in Example 1 as a 1.9 kb Eco RI-Xba I fragment and subcloned in a pUC19 vector previously digested with Eco RI and Xba I. .

The resulting 4.6 kb plasmid was defined as 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)

Underline of primer E indicates Bsp HI separation site and underline of primer F indicates Sma I or Xma I separation site.

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

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

Primer H: 5'-TGCTCTAGAAAGCTTAAGTTCACTGGTCGTCAGCGTCGAGGG-3 '

(SEQ ID NO: 12)

Underline in primer G indicates Bbs I separation site,

Underlined in primer H indicates the Af1 II separation site.

Primers E and F were used to amplify the fragment size 188bp in the 5 'region of the Lac5.5 cDNA gene by PCR reaction.

Primers G and H were used to amplify fragment size 110 bp in the 3 ′ region of the Lac5.5 cDNA gene.

100 μl PCR mixture contains 10 ng of Lac5.5 cDNA (in pBK CMV vector), 0.5 U of Tth polymerase, 1.25 mM of MgCl ₂ , 0.2 mM of 4 dNTPs each and a pair of primers E and F and a pair of primers G and 140 mol each of H was included.

The PCR reaction was carried out under the following conditions:

After 5 minutes at 94 ° C, 1 minute at 94 ° C, 2 minutes at 50 ° C, and 1 minute at 72 ° C, finally, 30 cycles of 7 minutes at 72 ° C were carried out.

In the PCR reaction, DNA fragments with primers E and F were first cleaved with Eco RI and Xma I, purified by gel electrophoresis, and cloned in vector pLac5 previously cleaved with Eco RI and Xma I.

As a result, a vector pLac51 obtained by introducing a BspHI separation site into a translation start codon (ATG) was obtained.

In the PCR reaction, DNA fragments with primers G and H were first cleaved with Eco RI and xba I, then purified by gel electrophoresis, and cloned in pUC19 vectors previously cleaved with Eco RI and xba I.

An insert of size about 100 bp was digested with Bbs I and Xba I in the resulting plasmid pLT5.

The Bbs I-xba I fragment was cloned in vector pLac51 finally digested with Bbs I and xba I.

As a result, a vector pLac 513 was obtained including a new Af1 II separation site at the 3 'end of the laccase cDNA gene.

The cDNA gene of Trametis versicolor laccase Lac5.5 with altered 5 'and 3' regions was isolated by BspHI by partial digestion of polamide pLac513.

As a result, a 2.6 kb fragment including the coding region of the Lac5.5 cDNA gene and about 1.1 kb of the pUC19 vector was obtained.

This fragment was cleaved with Af1 II in 2 steps, and then a Lac5.5cDNA fragment of 1.5 kb obtained was isolated.

This fragment was a vector pAN52 containing a size 4.0 kb fragment of the glaA promoter from Aspergillus niger, a size 0.7 kb fragment of the trpC transcription terminator obtained from Aspergillus nidulans and a size 2.7 kb fragment of the pUC18 vector. Ligation was carried out on Af1 II-Nco I fragments of size 7.4 kb at -12.

In pAN1acl, the glaA promoter region was functionally associated with the NTG I-Bsp HI junction to ATG detoxification codon in laccase cDNA gene.

B: Association of laccase Lac5.5 cDNA to glaA promoter by substitution of N-terminal signal sequence by glucoamylase fragment

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

Primer I: 5'-CCGGAATTCGATATCCAAGCGCGGGATCGGGCCTGTGCTCGAC-3 '

(SEQ ID NO: 13)

Underlined in primer I represents the Eco RV separation site.

To alter the 3 'region of the Lac 5.5 cDNA gene, privacy G and H were used (see Example A section of this).

Primer I and F were used to amplify fragment size 110 bp in the 5 'region of the Lac5.5 cDNA gene by PCR.

Primers G and H were used to amplify fragment size 110 bp in the 3 ′ region of the Lac5.5 cDNA gene.

PCR reactions were carried out as described in Example A.

In the PCR reaction, DNA fragments with primers I and F were digested with Eco RI and Xma I, purified by gel electrophoresis, and cloned in vector pLac5 previously digested with Eco RI and Xma I.

As a result, this was obtained in the vector pLac52 in which the Eco RV separation site by the amino acid codon of the array Ile Ser Lys Arg (SEQ ID NO: 14) was inserted in the 5 'region before the codon of the first amino acid of the mature laccase protein.

This arrangement is the recognition site of the KEX2 protease.

The 3 'region of the laccase cDNA gene in vector pLac52 was altered as described for vector pLac51.

In the PCR reaction, a size approximately 100 bp insert with primers G and H was isolated by cleavage with Bbs I and Xba I in plasmid pLT5.

The Bbs I-Xba I fragment was finally cloned in vector pLac52 digested with Bbs I and Xba I.

This resulted in a vector pLac523 containing a new AF1 II isolation site at the 3 'end of the laccase cDNA gene.

The cDNA gene of the Trametis versicolor laccase Lac5.5 with altered 5 'and 3' regions cleaves its plasmid pLac523 with Eco RV and Af1 II, resulting in a 1.5 kb Lac5. 5 cDNA fragments were isolated.

This fragment was ligated to an Af1 II-Eco RV fragment of size 9.3 kb in vector PAN56-9.

The vector pAN56-9 is a size 4.0 kb fragment of a glaA promoter obtained from Aspergillus niger, followed by a size 2.0 kb fragment coded for a glucoamylase fragment of Aspergillus niger, and 4 amino acids of the KEX2 separation site. A short DNA section encoded, a 0.7 kb fragment of trpC transcription terminator obtained from Aspergillus nidulans and a 2.7 kb fragment of pUC18 were included.

The Eco RV and Af1 II isolates arranged 3 ′ of the KEX2 isolate.

As a result, transformation in E. coli was obtained in a size 10.8 kb vector of PANlac2.

In PANlac2, a coding region of the cDNA gene of mature laccase Lac5.5 is first expressed so that a fragment of glucoamylase containing a signal sequence and a recognition sequence of KEX2 protease and a molten protein consisting of mature laccase Lac5.5 are produced. Linked to the coding region of the glucoamylase gene.

Upon secretion, this molten protein was isolated by KEX2 protease and the mature laccase was secreted into the culture synergist.

C: binding of amdS and pyrG markers to the vectors pANlac1 and pANlac2

5 μg of each vector was digested with NOT I overnight to linearize pANlac 1 and pANlac2.

Subsequently, they were treated with calf intestinal alkaline phosphatase, phenol / chloroform extract and ethanol precipitates of mammalian pups.

The genes of the selectable markers amdS (acetamidase) and pyrG (orotidine-5'-monophosphate decarboxylase) are present in polamide pAN52-11, digested with NOT I, and then sized to 6.4 kb. It could be separated as a fragment.

E. coli JM109 was transformed by ligation into 0.6 μg of Not I fragment, which was linearized and treated with phosphatase treated vectors pANlac 1 and pANlac2, respectively.

The polamide DNAs were prepared in ampicillin-resistant colonies obtained from their transformation, cleaved with NOT I and analyzed by agarose gel electrophoresis.

Six of the eight analytical clones obtained from transformation with the vector pANlac1 and five of seven analytical clones obtained from the transformation with the vector pANlac 2 contained a 6.4 kb gene NOT I fragment.

The vectors with the selectable marker gene were set to pANlaclS (size 15.3 kb, FIG. 1) and pANlac2S (size 17.2 kb, FIG. 2).

Three of the six obtained pANlac1S clones contained a Not I fragment at the required orientation shown in FIG. 1 and one of the five obtained pANlac2S clones included a Not I fragment at the required orientation shown in FIG.

Example 7

Transformation of Aspergillus

Strains Aspergillus niger AB1.13 (pryG ^_ ) (W. van Hartingsveldt, IE Mattern, CMJ van Zeij1, PH pouwels and CAMJJ van den Hondel (1987) Mol. Gen. Genet. 206, 71-75) and As Pergillus awamori (strain ATCC 11358) was used for the transformation.

The Aspergillus transformation was carried out by 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 micelle rate with Novozym 234.

Newly prepared by disintegrating the mycelium obtained from one flask in a sterile Erlenmeyer flask, dissolving the lytic enzyme mixture Novozym 234 (Novo Nordisk) in OM medium (0.27 M calcium chloride, 0.6 M NaCl). Suspended in 15 ml of one solution.

The micelles resuspended in the enzyme solution were incubated at 30 ° C. for 1 to 3 hours at a slow speed (80 rpm).

When incubating, the production of the protoplasts was observed under a microscope.

Freely moving protoplasts were generally observed after 1 hour.

After obtaining a large number of freely movable protoplasts, these protoplasts were separated from the remaining micelles by filtration through a glass filter, Miracloth (commercial calbiochem) and carefully removed STC broth (1.2M sorbitol). , 50 mM calcium chloride, 35 mM NaCl, 10 mM Tris-HCl, pH7.5).

Protoplasts were separated by centrifugation of the suspension in a sterile sample vessel (2000 rpm, 4 ° C., 10 minutes) and washed twice with STC medium.

The concentration of the protoplasts was measured under a microscope in the counting chamber.

The protoplast suspension was adjusted to concentration 1 × 10 ⁸ protoplasts / ml in protoplast regeneration and transformation experiments.

Protoplasts in Aspergillus niger and Aspergillus awamori were transformed with plasmids pANlac1S and pANlac2S.

The two protoplasts included the pyrG gene (code of orotidine-5'-phosphate decarboxylase) and the amdS gene (code of acetamylase) as select markers.

An aliquot of 0.1 ml of protoplasts was mixed with 10 µg of plasmid DNA in a 12 ml culture vessel and incubated on ice for 25 minutes.

Then, 1.25 mL of 60% PEG 4000 solution (60% PEG4000, 50 mM calcium chloride, 10 mM tris-HCl, pH7.5) was slowly added to the transformed mixture with repeated mixing.

After further incubating at 20 ° C. for 20 minutes, the reaction vessel was filled with STC medium solution, mixed at 4 ° C. for 10 minutes, and centrifuged.

The pellet was resuspended and coated in an osmotic selective medium stabilized with sorbitol.

The plate was incubated at 30 ° C. for 7 days to check the growth of colonies.

In several trials, the transformation rate was transformant 1-5 per μg of Polismid DNA of Aspergillus niger and 0.1-0.5 transformant per μg of Plasmid DNA of Aspergillus awamori.

Transformants were transferred to selection plates and purified with acetamide.

Transformants with high copy numbers were identified by coating with acrylamide on a selection plate.

Acrylamide is a poor substrate of the acetamilase enzyme encoded by the amdS gene as compared to acetamide, and can only help growth with transformants with a high copy number of the amdS gene.

Transformants capable of functional expression of the laccase enzymes were identified by coating these transformants with plates of malAdextrin, an expression inducer of glaA promoter, and growing at 28 ° C. for 2 days. And ABTS agar (0.1% ABTS, 1% agarose, pH 4.5 in McIllvaine buffer solution).

The laccase-expressing transformants were shown by green halo formation.

Spore suspensions were prepared in transformants, which showed good growth on acrylamide plates and showed positive reaction with ABTS in the activity test.

Example 8

Expression of Trametis Bus-Collar Lacase Lac5.5 in Aspergillus

Expression of laccase Lac5.5 in Aspergillus was examined on a shaken flask scale.

The following medium was used:

5% (w / v) solution of maltodextrin dissolved in tap water was treated in an autoclave for 20 minutes.

To 500 ml of this base broth was added 1 ml of 1M magnesium sulfate, 0.5 ml of 1000 × trace element solution, 10 ml of 50 × AspA solution and 5 ml of 10% (w / v) casamino acid.

The 100 x trace element solution has the following composition:

ZnSO ₄ x 7H ₂ O 2.2 g,

H ₃ BO ₃ 1.1 g,

0.5 g MnCl ₂ x 4H ₂ O,

0.5 g FeSO ₄ x 7H ₂ O,

0.17 g of CoCl ₂ x 5H ₂ O,

CuSO ₄ x 5H ₂ O 0.16 g,

0.15 g Na ₂ MoO ₄ x 2H ₂ O and

5 g of EDTA was dissolved in 80 ml of H ₂ O.

The 50 x AspA solution has the following composition:

150 g of NaNO ₃ , 13 g of KCl, and 38 g of KH ₂ PO ₄ were dissolved in 500 mL of H ₂ O and adjusted to pH5.5 using 10M KOH.

CuSO ₄ x 5H ₂ O was further added to the medium solution of the final concentration of 0.5 mM.

For expression experiments, 50 ml of culture medium in a 300 ml Erenmeyer flask was incubated with 1 × 10 ⁶ spores / ml.

Cultivation was performed at 300 ° C. at 30 ° C. in a shaker incubator.

The samples were taken out for one week and the laccase activity of the culture supernatant was measured. The maximum laccase activity was observed for 60 to 100 hours of growth, and the activity was 0.5 to 2.5 U / ml.

The laccase activity was measured in a McIllvaine buffer (PH4.5, temperature 37 ° C.) using a baseline ABTS (analytical value 0.1 mM) by colorimetry.

Makil vane buffer solution was prepared by titrating 0.1 M citric acid solution to pH 4.5 with respect to 0.2 M Na ₂ HPO ₄ solution.

The absorbance increase was measured at 420 nm (absorption coefficient of ABTS at 420 nm: 3.6 x 10 ⁴ 1 x mol ^-1 x cm ^-1 ).

Lactase activity 1U was defined as 1 mol of ABTS substrate converted per minute.

Example 9

Expression of Trametis Versicolor Lacquer Lac 5.5 in pichia pastoris

An expression system commercially available from Invitrogen was used in conjunction with its associated expression vectors (pPIC3 and pPIC9) and Pachiapastoris strains (GS115 and KM71).

The Pachia Pastoris strains GS115 and KM71 are histidine- auxotrophic.

The expression vector contained the terminator and promoter of the alcohol oxidase gene AOX1 obtained from Pichia pastoris.

The cDNA gene of Trametis versicolaclacase Lac5.5 was cloned between their two genetically regulated components.

The vector pP1C9 contains a DNA sequence coded for the signal sequence of an alpha-factor protein obtained from Saccharomyces cerevisiae downstream of the positioned AOX1 promoter, followed by the recognition sequence Glu-Lys. A short DNA section encoded for -Arg-Glu-Ala-Glu-Ala (SEQ ID NO: 15) was included.

The vector contained an ampicillin-resistant gene for selection in E. coli.

The vector contained the HIS4 gene from Pichia pastoris for selection of Pichia pastoris.

A: Structure of laccase Lac5.5 expression vector having a signal sequence of laccase Lac5.5

Polismid pLac5.5 and primers K and L were used for amplification of the laccase enzyme.

Primers F and G had the following arrangement:

Primer K:

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

Primer L:

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

Primer K included an array of Eco RI cleavage sites (underlined) followed by a DNA sequence for the first seven amino acids of the laccase Lac5.5 signal sequence.

Primer L contains a sequence of Not I isolation sites followed by a translation stop codon at the complementary and reverse orientation and the DNA sequence for the last 7 amino acids of laccase Lac5.5. It included.

PCR amplification was carried out by conventional techniques known to those skilled in the art.

20 ng of pLac5.5 DNA was used in a 50 μl PCR reaction containing 0.5 U of Vent polymerase, 1 mM MgCl ₂ , 4 dNTPs each 0.2 mM, primer K and L 100 pmol each.

Other conditions for the desired amplification of the required PCR product are as follows:

25 cycles of 5 minutes at 94 ° C, then 1 minute at 94 ° C, 1 minute at 55 ° C and 2 minutes at 72 ° C.

PCR fragments with the expected size of 1.5 kb were obtained.

The PCR fragment was purified by agarose gel electrophoresis, digested with restriction enzymes Eco R1 and Not I, precipitated with ethanol and taken up in H ₂ O.

The vector pPIC3 was digested with Eco RI and Not I, purified by agarose gel electrophoresis and separated and treated with alkaline phosphatase.

Then, the mixture was extracted with phenol / chloroform (ratio of 3: 1) and subjected to ethanol precipitation.

The DNA fragment of laccase Lac5.5 prepared by PCR amplification was ligated to the pPIC3 vector thus prepared, and E. coli top 10F 'cells (Invitrogen) were transformed therewith.

Plasmid DNA was isolated from ampicillin-resistant clones and cloned 1.5 kb inserts were identified by restriction digestion with EcoRI and Not I.

Nine of the 12 clones examined were positive.

The vector thus obtained is represented by the name pL512 (FIG. 3).

B: Composition of laccase Lac5.5 expression vector with its alpha-factor signal sequence in Saccharomyces cerevisiae

Lactase gene was heavy using plasmid pLac5.5 and primers L and M.

Primer M had the following arrangement:

Primer M:

5'-ACTCGAGAATTCGGGATCGGGCCTGTGCTCGACCTCACG-3 '(SEQ ID NO: 18)

Primer M included an array of Eco R1 cleavage sites (underlined), followed by DNA sequences for the first nine amino acids of the putative N-terminus of laccase Lac5.5 which were then processed.

The N-terminus of the laccase Lac5.5 that did the processing was estimated in comparison with other laccase arrangements.

DNA fragments for the processing form of the laccase Lac5.5 were prepared by PCR amplification using pLac5.5 cDNA and primers L and M as described in section A of this Example.

The vector pPIC9 was cleaved with EcoRI and Not I, ligated to the DNA fragment of laccase Lac5.5 prepared by the pPIC3 vector described in Section A of this Example, and prepared by PCR amplification. Cells (Invitrogen) were transformed with them.

The plasmid DNA was isolated into ampicillin-resistant clones and the cloned 1.5 kb insert was identified by restriction digestion with Eco RI and Not I.

Three of the 12 clones examined were positive.

The vector thus obtained is represented by the name pL532 (FIG. 4).

C: Transformation of Pichia pastoris

Pichia pastoris strains GS115 and KM71 were first incubated overnight at 30 ° C. in 5 ml of YPD medium (1% yeast extract, 2% peptone, 2% dextrose).

Two main cultures were inoculated into each 250 ml of YPD medium using 0.2 ml of the preculture, and the culture was again incubated overnight at 30 ° C. until the light concentration (OD600 nm) was 1.3-1.5.

The yeast cells obtained from 250 ml of main culture were then centrifuged (1500xg for 5 minutes), washed twice with 200 ml of H ₂ O per hour and once with 10 ml of 1 M sorbitol, and finally in 0.5 ml of 1 M sorbitol. Absorbed.

Plasmid DNA of the vector pL512 or pL532 was linearized with Stu 1 or Nsi I, precipitated with ethanol and taken up in H ₂ O at a concentration of 1 μl of DNA per μl.

The transformation mixture contained 80 µl of pichia pastoris cells and 10 µg of linearized vector DNA.

Transformation was performed by electroporation at 1500V, 25 Hz and 200 Ohm (BioRad Gene Pulser).

The discharge time was about 4.2 msec.

1 ml of 1M sorbitol was added to the transfection mixture, which was incubated on ice for 30 minutes and then histidine-free MD plate (1.34% YNB, yeast substrate, 4 x 10 ^-5 biotin, 1% dextrose, 1.5 % Agar) in 0.3 ml aliquots.

The transformants appeared after 3-5 days of incubation at 30 ° C.

Transformants were purified by two striaking inoculations on MD plates.

Lackase producers were identified on MM indicator plates.

MM indicator plates included 1.34% YNB, 4 × 10 ⁻⁵ % biotin, 0.5% methanol, 1.5% agar, 1 mM ABTS and 0.1 mM Cu sulfate.

Inducer metanole was set in the lid of the Petri dish and supplemented daily so that colonies could be fed by diffusion into the methanol.

Lacquer makers incubated for 2-3 days at 30 ° C. and then produced green halo.

E. Expression in shake flasks

Lactase-producing Pichia pastoris transformation in 50 ml of EMGY medium (1% yeast extract, 2% peptone, 0.1mK phosphate, pH6.0, 1.34% YNB, 4 × 10 ^-5 % biotin, 1% glycerol) The inoculation was incubated in a shaker for 48 hours at 300rpm and 28 ℃.

The cells obtained in this pre-culture was separated by centrifugation (1500xg, 10 minutes), the main culture medium of 10㎖ (MMY, 1% yeast extract, 2% peptone, 1.34YNB, 4 x 10 ^-5% biotin, 3% methanol).

The main culture medium was further incubated at 300 rpm and room temperature in a shaker.

The main culture medium was supplemented with methanol (0.3 ml / 10 ml) at intervals of 24 hours.

Production of recombinant laccase Lac5.5 started after 24 hours.

Production rates up to 4 U / mL were obtained 190 hours after the start of the main culture medium.

Example 10

Isolation of Recombinant Lacase Lac5.5

Recombinant laccase Lac5.5 was obtained by culturing the transformed Aspergillus strain in shake flasks as described in Example 8.

Culture supernatants containing laccase Lac5.5 were concentrated by cross-flow filtration.

Sartocon microfiltration modules (Sartorius) with an exclusion limit of 30 KD were used here.

The concentrated laccase Lac5.5 was then dissolved by lyophilization in 20 mM Naphosphate at pH 6.0.

The concentrated recombinant laccase Lac5.5 had an activity of 18.6 U / ml.

The laccases concentrated by cross flow filtration were dialyzed against pH6.5, 20 mM bistris-HCl.

Next, the conductivity 1.5 mS / cm was measured.

Dialyzed laccase was then treated by chromatography on a column of DEAE-Sepharose (Pharmacia) equilibrated at pH6.5 and 20 mM bistris-HCl (load buffer solution).

Under this condition, the laccase binds to DEAE-Sepharose.

When eluted with a linear gadient of 0-0.5 M NaCl in the load buffer solution, the laccase activity was restored at a salt concentration of 0.15 M NaCl.

The laccase fraction on the DEAE-Sepharose column was mixed with 20% saturated ammonium sulfate and pH 4.5 (final concentration 20 mM) of Na acetate.

The pH was adjusted to pH 4.5 with acetic acid.

The laccase fraction thus prepared was then chromatographed on a column of phenyl-sepharose pharmacia equilibrated in a load buffer solution (20 mM Naacetate, pH4.5, 20% ammonium sulfate).

In this state, laccase bound to phenyl-sepharose.

The laccase activity was recovered at 16% ammonium sulfate saturation when eluted with a linear regime of 20-0% saturated ammonium sulfate in pH 4.5, 20 mM Na acetate.

The laccase fraction was dialyzed against pH6.5, 20 mM bistris-HCl, bound to DEAE-Sepharose, concentrated, and eluted with 0.3 M NaCl.

Elution with 0.3M NaCl and binding were performed at pH6.5 and 20mM bistris-HCl, respectively.

The yield of laccase activity based on the starting material was 20%.

The isolated laccase was analyzed by N-terminal amino acid sequence.

The arrangement determined by this analysis, Gly Ile Gly Pro Val Leu Asp Leu Thr Ile Ser Arg Ala Val (SEQ ID NO: 19), was consistent with the N-terminus of mature laccase Lac 5.5 derived from cDNA sequence and homology comparison.

Example 11

Biochemical Properties of Recombinant Lacase Lac5.5

As described in Example 8, the optimum pH and temperature and stable pH and temperature of the recombinant laccase Lac5.5 prepared by Aspergillus were investigated.

In this experiment, recombinant laccase Lac5.5 buffer was first replaced with a Sepildex buffer of pH4.5 in Sephadex G25 (Pharmacia, PD10 column).

A: optimum pH

Suitable buffer solutions for each of the pH values shown in FIG. 5 were prepared by suitably mixing the unbuffered Na citrate solution with the Na phosphate solution.

Then, the laccase activity of recombinant laccase Lac5.5 was at 37 ° C. It was measured at each pH.

As shown in FIG. 5, laccase Lac5.5 has optimal activity in the strongly acidic range of the base ABTS.

B: pH stability

Lacase Lac5.5 was pre-cultured in McIllbain buffer at pH 3.0, 4.5 and 6.0 (temperature 37 ° C.).

Aliquots were taken after 0, 30, 60 and 120 minutes and diluted in membrane ilvane buffer at pH4.5.

The laccase activity was not adversely affected by pretreatment at pH4.5 and 6.0.

The half-life of laccase at pH 3.0 was between 60 and 120 minutes (FIG. 6).

C: optimum temperature

The laccase activity of the recombinant laccase Lac5.5 was measured at the temperature shown in FIG.

The laccase activity was measured using ABTS assay in membrane ilvane buffer at pH4.5.

The optimal temperature of laccase Lac5.5 was 70 ° C, which was higher than expected in this activity measurement.

The temperature when the measured laccase activity was half of the maximum was 50 ° C and 80 ° C.

D: Temperature Stability

Lacase Lac5.5 was pre-cultured in membrane ilvane buffer at pH 4.5 at temperatures 45, 55 and 65 ° C.

Aliquots were taken after 0, 30, 60 and 120 minutes and diluted in McIllvane buffer at pH4.5.

The laccase activity was measured at 37 ° C.

The laccase activity was not adversely affected by pretreatment at 45 ° C.

Measurements after preincubation at 55 ° C. for 120 minutes indicated 80% of the remaining activity.

The laccase half life at 65 ° C. was 60 minutes (FIG. 8).

Array list

(1) General Information

(i) Applicant

(A) Full Name: Consodium Wheate Electrohemiche Instituri GmbH

(B) Alias: Gielstad Strasse 20

(C) City Name: Munich

(E) Country name: Federal Republic of Germany

(F) Zip code: De-81379

(G) Phone: 089 74844 0

(H) Fax number: 089 74844 350

(ii) Application name: DNA sequence, expression of the DNA sequence, encoded by the DNA sequence

Thermophilic laccase and its use

(iii) Array Number: 19

(iv) computer-readable form

(A) Intermediate type: floppy disk

(B) Computer: IBM PC Compatible

(C) Operating System: PC-DOS / MS-DOS

(D) Software: Patent In Release # 1.0, Version # 1.25 (EPO)

(2) SEQ ID NO: 1, Information:

(i) Array characteristics:

(A) Length: 1572 base pairs

(B) type: nucleic acid

(C) strandedness: single

(D) Topology: Shipboard

(ii) Molecular type: cDNS to mRNS

(iii) Hypothetical: None

(iii) antisense: NO

(vi) original source:

(A) Organisms trametis versicolor: (Trametes Versicolor)

(B) strain: TV-1

(vii) immediate source:

(B) clone: plac55

(xi) Array description: SEQ ID NO: 1:

(2) Information of SEQ ID NO: 2:

(i) Array characteristics:

(A) Length: 1572 base pairs

(B) type: nucleic acid

(C) strandedness: single

(D) Topology: Shipboard

(ii) Molecular type: cDNS to mRNS

(iii) High Potential: None

(iii) Antisense: None

(vi) Origin:

(A) Creature: Tramethis versicola

(B) strain: TV-1

(vii) direct employees:

(B) clone: plac55

(xi) Array description: SEQ ID NO: 2:

(2) Information of SEQ ID NO: 3:

(i) Array characteristics:

(A) Length: 524 amino acids

(B) Type: Amino Acid

(D) Topology: Shipboard

(ii) Molecular Type: Protein

(iii) High Potential: Yes

(vi) Origin:

(A) Creature: Tramethis versicola

(B) strain: TV-1

(ix) Features:

(A) Name / Key: Protein

(B) Location: 1..524

(xi) Array description: SEQ ID NO: 3:

(2) Information of SEQ ID NO: 4:

(i) Array Features:

(A) Length: 17 base pairs

(B) type: nucleic acid

(C) Trendedness: Single

(D) Topology: Shipboard

(ii) Molecular Type: cDNS

(iii) High Potential: Yes

(iii) Antisense: None

(xi) Array description: SEQ ID NO: 4:

TGGCAYGGNT TYTTYCA 17

(2) Information of SEQ ID NO: 5:

(i) Array characteristics:

(A) Length: 14 base pairs

(B) type: nucleic acid

(C) Trendedness: Single

(D) Topology: Shipboard

(ii) Molecular Type: cDNS

(iii) High Potential: Yes

(iii) Antisense: None

(xi) Molecular description: SEQ ID NO: 5:

TCDATRTGRC ARTG 14

(2) Information of SEQ ID NO: 6:

(i) Array Features:

(A) Length: 27 base pairs

(B) type: nucleic acid

(C) Trendedness: Single

(D) Topology: Shipboard

(ii) Molecular Type: cDNS

(iii) High Potential: Yes

(iii) Antisense: None

(xi) Array description: SEQ ID NO: 6:

ATTCAGGGAT CCTGGTAYCA YWSNCAY 27

(2) Information of SEQ ID NO: 7:

(i) Array Features:

(A) Length: 27 base pairs

(B) type: nucleic acid

(C) Trendedness: Single

(D) Topology: Shipboard

(ii) Molecular Type: cDNS

(iii) High Potential: Yes

(iii) Antisense: None

(xi) Array description: SEQ ID NO: 7:

ATACGAGGAT CCRTGNCCRT GNARRTG 27

(2) SEQ ID NO: 8, Information:

(i) Array Features:

(A) Length: 3284 base pairs

(B) type: nucleic acid

(C) Trendedness: Single

(D) Topology: Shipboard

(ii) Molecular Type: DNS Genome

(iii) High Potential: None

(iii) Antisense: None

(vi) Origin:

(A) Creature: Tramethisversikol

(B) strain: TV-1

(vii) direct employees:

(B) clone: plac56chr

(xi) Array description: SEQ ID NO: 8:

(2) Information of SEQ ID NO: 9:

(i) Array Features:

(A) Length: 39 base pairs

(B) type: nucleic acid

(C) Trendedness: Single

(D) Topology: Shipboard

(ii) Molecular Type: cDNS

(iii) High Potential: Yes

(iii) Antisense: None

(xi) Molecular description: SEQ ID NO: 9:

CCGGAATTCA TGACTGGGCT GCGTCTCCTT CCTTCCTTC 39

(2) SEQ ID NO: 10's Information:

(i) Array Features:

(A) Length: 19 base pairs

(B) type: nucleic acid

(C) Trendedness: Single

(D) Topology: Shipboard

(ii) Molecular Type: cDNS

(iii) High Potential: Yes

(iii) Antisense: None

(xi) Array description: SEQ ID NO: 10:

GAGAGGCCCG GGAGCCTGG 19

(2) Information of SEQ ID NO: 11:

(i) Array Features:

(A) Length: 31 base pairs

(B) type: nucleic acid

(C) Trendedness: Single

(D) Topology: Shipboard

(ii) Molecular Type: cDNS

(iii) High Potential: Yes

(iii) Antisense: None

(xi) Array description: SEQ ID NO: 11:

GCTGAATTCG AAGACATCCC CGACACCAAG G 31

(2) SEQ ID NO: 12's Information:

(i) Array Features:

(A) Length: 42 base pairs

(B) type: nucleic acid

(C) Trendedness: Single

(D) Topology: Shipboard

(ii) Molecular Type: cDNS

(iii) High Potential: Yes

(iii) Antisense: None

(xi) Array description: SEQ ID NO: 12:

TGCTCTAGAA AGCTTAAGTT CACTGGTCGT CAGCGTCGAG GG 42

(2) Information of SEQ ID NO: 13:

(i) Array Features:

(A) Length: 43 base pairs

(B) type: nucleic acid

(C) Trendedness: Single

(D) Topology: Shipboard

(ii) Molecular Type: cDNS

(iii) High Potential: None

(iii) Antisense: None

(xi) Array description: SEQ ID NO: 13:

CCGGAATTCG ATATCCAAGC GCGGGATCGG GCCTGTGCTC GAC 43

(2) Information of SEQ ID NO: 14:

(i) Array Features:

(A) Length: 4 amino acids

(B) Type: Amino Acid

(D) Topology: Shipboard

(ii) Molecular Type: Peptide

(iii) High Potential: Yes

(v) Short piece type: Internal

(vi) Origin:

(A) Creature: Aspergillus niger

(xi) Array description: SEQ ID NO: 14:

(2) SEQ ID NO: 15, Information:

(i) Array Features:

(A) Length: 7 amino acids

(B) Type: Amino Acid

(D) Topology: Shipboard

(ii) Molecular Type: Peptide

(iii) High Potential: Yes

(v) Short piece type: Internal

(vi) Origin:

(A) Creature: Saccharomyces cerevisiae

(xi) Array description: SEQ ID NO: 15:

(2) SEQ ID NO: 16, Information:

(i) Array Features:

(A) Length: 38 base pairs

(B) type: nucleic acid

(C) Trendedness: Single

(D) Topology: Shipboard

(ii) Molecular Type: cDNS

(iii) High Potential: Yes

(v) Antisense: None

(xi) Array description: SEQ ID NO: 16:

ACTCGAGAAT TCACCATGAC TGGGCTGCGT CTTCTTCC 38

(2) Information of SEQ ID NO: 17:

(i) Array Features:

(A) Length: 41 base pairs

(B) type: nucleic acid

(C) Trendedness: Single

(D) Topology: Shipboard

(ii) Molecular Type: cDNS

(iii) High Potential: Yes

(v) Antisense: None

(xi) Array description: SEQ ID NO: 17:

ACTAGAGCGG CCGCCTATCA CTGGTCGTCA GCGTCGAGGGC 41

(2) SEQ ID NO: 18, Information:

(i) Array Features:

(A) Length: 39 base pairs

(B) type: nucleic acid

(C) Trendedness: Single

(D) Topology: Shipboard

(ii) Molecular Type: cDNS

(iii) High Potential: Yes

(v) Antisense: None

(xi) Array description: SEQ ID NO: 18:

ACTCGAGAAT TCGGGATCGG GCCTGTGCTC GACCTCACG 39

(2) Info of SEQ ID NO: 19:

(i) Array Features:

(A) Length: 14 amino acids

(B) Type: Amino Acid

(D) Topology: Shipboard

(ii) Molecular Type: Peptide

(iii) High Potential: None

(v) Short piece type: N-terminal

(xi) Array description: SEQ ID NO: 19:

The present invention codes for a protein having laccase activity, and comprises a DNA sequence consisting of SEQ ID NO: 1 from SEQ ID NO: 76 to position 1572 or SEQ ID NO: 2 from position 76 to position 1572 and a DNA sequence thereof. It relates to a DNA sequence having more than 80% sequence homology.

SEQ ID NO: 1 and SEQ ID NO: 2 show DNA sequences encoding positions 1 to 75 for signal sequence for secretion of proteins.

This signal sequence can be replaced by another signal sequence for protein secretion.

The new DNA sequence can be obtained, for example, by cloning from the Basidiomycetes strain, Trametis versicolor TV-1 (deposited in DSMZ, Accession No. 11523).

For this acquisition, a gene bank was set from Trametisversicollar TV-1 obtained by a known method.

This is a cDNA or genomic gene bank.

To isolate new DNA sequences in this genebank, DNA probes containing laccase-specific DNA sequences were used.

This type of DNA probe can be obtained, for example, by PCR using DNA primers from the genomic DNA of Tramethisversacler TV-1.

The primers used are degenerate DNA sections, preferably 14-27 bp in length, and the DNA is established by comparison with an array of known laccase genes.

It is preferable that the DNA section suitable as a primer is obtained by oligonucleotide synthesis of the already established DNA section.

New laccase enzymes can be isolated as described in Examples 1-5, for example.

Thus, the laccaas gene isolated by the Example can be modified by the technique known to a person skilled in the art.

For example, at any preferred position in the configuration, modifications can be made by site directed mutagenesis.

Accordingly, the present invention also encodes for a protein having laccase activity, wherein the DNA sequence comprises SEQ ID NO: 1 at positions 76 to 1572 or SEQ ID NO: 2 at positions 76 to 15157 for at least 80% homology. It relates to a DNA sequence consisting of a DNA sequence having a sequence homology.

In order to express the new DNA, the latter is cloned in an expression vector by known methods.

An expression vector containing the laccase gene is introduced into a microorganism and expressed in the microorganism.

The expression vector can be a DNA structure that binds to the genome of a host organism and replicates with the organism.

The expression vector may be, for example, an autoreplicating DNA structure that does not bind to the host genome, such as a plasmid, artificial chromosome or contrasting extra chromosomal genetic element.

Suitable expression vectors preferably include the following genetic elements.

A promoter that promotes the expression of the laccase gene in the host organism, which is preferably a strong promoter in order to increase the expression efficiency.

This promoter is preferably functionally bound to the 5 'end of the laccase gene.

Suitable and accelerators are selected from the group of tac promoters, subtilisin promoters, GAL promoters, TAKA arylase promoters, polyhedrin promoters, glucoamylase promoters, gapDH promoters and alcohol oxydiaze promoters.

The tac promoter is suitable and preferred for expression in E. coli, the subtilisin promoter for expression in Bacillus, the GAL promoter for the yeast Saccharomyces cerevisiae (Saccharomyces) cerevisiae), its TAKA amylase promoter is expressed in Aspergillus niger, its polyhedrin promoter, or its glucoamylase promoter or its yeast in Aspergillus niger. Alcohol oxidase promoters in pichia pastoris are each suitable and preferred for expression in baculovirus-infected insect cells.

Glucoamylase promoters of Aspergillus niger or alcohol oxidase promoters of yeast Pichia pastoris are particularly suitable for the expression of new thermophilic laccases.

The expression vector is also suitable for host organisms, for transcription termination, and for polyadenylation in eukaryotes that need to functionally bind to the 3 'end of the laccase gene. It is preferable to include an additional signal.

The expression protein is preferably secreted into the medium by the host organism.

The secretions produced by the host organism are formed by the N-terminal signal sequence.

The signal sequence is a natural signal sequence present in the laccase gene or a heterologous signal sequence in which a coding DNA functionally binds to the 5 'end of the laccase gene in an expression vector.

The signal sequence of the following secreted proteins is preferred:

Alpha-cyclodextrin glucosyl transferase in Klebsiella oxytoca, subtilisin in Bacillus subtilis, in Saccharomyces cerevisiae Alpha-factor, phosphatase in pinchia pastoris, alpha-amylase in Aspergillus niger, glucocorticoid in Aspergillus niger or Aspergillus awamori Signal sequences naturally present in amylase or laccase enzymes.

Signal sequences that are naturally present in the laccase gene are particularly suitable and include the following nonhomologous signal sequences:

Signaling arrangement of glucoamylase in Aspergillus niger or Aspergillus awamori, alpha-factor in Saccharomyces cerevisiae or phosphatase in Pichia pastoris.

The laccase secretion can also be obtained by expression of a fusion protein.

Here, the secreted fragment or secreted protein gene of this protein is functionally linked to the laccase gene in the expression vector.

In this connection, the expression of fusion proteins consisting of N-terminal fragments of glucoamylase and thermophilic laccase in Aspergillus niger is particularly preferred.

In addition, it is preferable to select the point of attachment between the glucoamylase and laccase.

Therefore, the amino acid sequence of this binding point acts as a recognition site of the peptidase to be processed in the secretory mechanism of the host cell, so that the expression fusion protein is degraded in vivo and the laccase is released.

Moreover, it is preferable that the expression vector contains the gene of a selection marker.

The selection marker encoded by the gene can confer resistance to the antibiotic of the host organism or compensate for the defect of the host organism.

Preferred selection markers are genes that are resistant to antibiotics such as ampicillin, kanamycin, chloramphenicol, tetracycline, hygromycin, zeosin or bialaphos.

Preferred selection markers to compensate for growth defects are genes such as amdS, pyrG, trpC, His4, niaD, argB or hygB.

As selection markers, genes resistant to the amdS and pyrG genes and to his4 and the antibiotic zeocin are particularly suitable.

In addition, the selection marker gene may exist together with the laccase gene in one DNA molecule, and the two genes may exist separately in different DNA molecules.

In the latter case, the host organism undergoes cotransformation with two DNA molecules.

In this way, the present invention also relates to an expression vector constituting a new DNA sequence.

Suitable and preferred microorganisms expressing the new expression vectors include microorganisms of bacterial origin such as E. coli or Bacillus subtilis, yeasts of Saccharomyces or Pichia, or other Aspergillus. There are microorganisms of eukaryotic origin such as filamentous fungi of genus Rus, Trichoderma, Neurospora or Schyzophillum or culture of eukaryotic cells such as baculovirus-infected insect cells.

Especially preferred are filamentous fungi of the genus Aspergillus, such as Aspergillus niger or Aspergillus awamori, or yeasts such as Saccharomyces cerevisiae or Pichia pastoris.

Proteins encoded by the new DNA have the following biochemical properties:

The protein has the enzymatic activity of laccase.

The optimum pH of the enzyme activity is in the acidic range, has a peak pH of 2.0, and a pH of 4.0 for a half-maximal enzyme activity.

Enzyme stability at a temperature of 45 ° C. and pH 4.5-6.0 is 100% for a period up to 2 hours.

The enzyme stability at a temperature of 45 ° C. and pH 3.0 was 50% for a period of up to 1 hour, and the optimum temperature at pH 4.5 was 70 ° C.

Enzyme activity at the temperature of 65'-75 degreeC is 80% of the maximum activity.

The enzyme activity at a temperature of 50 ° C to 80 ° C is 50% of the maximum activity.

Enzyme stability between pH4.5 and temperature 55 ° C is 90% for a period up to 2 hours.

The enzyme stability at pH 4.5 and at 65 ° C. is 50% for a period of up to 1 hour.

The new protein consists of the protein sequence SEQ ID NO: 3.

The new protein is preferably produced by expressing a new DNA sequence in the microorganism described above.

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

As such, the present invention relates to a microorganism consisting of a new DNA sequence or a new expression vector.

Particular preference is given to using a combination of microorganisms and expression systems which allow them to secrete their proteins.

Such preferred combinations are as follows:

Use of glucoamylase to express its new DNA sequence in Aspergillus niger or Astermoris awamori

The preferred secretion signal used in this expression system is the thermophilic laccase itself or the signal sequence of the glucoamylase portion of the glucoamylase-laccase melt protein.

Use of Alcohol Oxidase Promoters Expressing New DNA Sequences in Pichia pastoris

The secretion signal used in this expression system is preferably a signal sequence of thermophilic laccase itself or an alpha-factor signal sequence in Saccharomyces cerevisiae or a phosphatase signal sequence in Pichia pastoris.

The new protein is suitable for all uses known as laccases.

These proteins are particularly suitable for delignification of pulp and depolymerization of polymer assemblies.

Lacase is also used for the deinking of waste paper, the polymerization of aromatic compounds in wastewater treatment, in particular for the polymerization of lignin-containing wastewater from pulp bleaching or for detoxification of contaminated soil.

The field of use also relates to the activation of dyes by oxidation of the dyes and reaction with precursor components which produce pigments.

Areas of use in organic synthesis include coupling reactions of aromatic compounds or oxidation of substituents of aromatic compounds, and in this connection, for example, oxidation of benzyl alcohols to their corresponding aldehydes, which avoids further oxidation of carboxylic acids. have.

In the uses described above, laccases can use the laccases themselves in their associated reactions, or else in combination with mediators.

Examples of such modifiers are ABTS or N-hydroxybenzotriazole.

As such, the present invention also relates to degreasing of pulp, depolymerization of polymer aggregates, deinking of waste paper, waste water, especially polymerization of aromatic compounds in lignin-containing wastewater obtained from pulse bleaching, oxidation of dyes, and activation of dyes to form pigments. And the use of new proteins for the coupling reaction of aromatic compounds in organic synthesis or for the side chain oxidation of aromatic compounds.

The use described above can be carried out using the new laccase protein itself or in combination with a promoter.

A new protein is obtained by the present invention, the new protein can be deliginized of pulp, depolymerization of polymer aggregates, deinked waste paper, polymerization of aromatic compounds in lignin-containing wastewater obtained from pulp bleaching as wastewater, oxidation of dyes, It can be used for activation of dyes forming pigments, coupling reactions of aromatic compounds in organic synthesis, or branched oxidation of antiaromatic compounds.

It can also be used to detoxify contaminated soil.

This new protein is suitable for all applications known as laccases.

Claims (10)

In the DNA sequence encoding the protein having laccase activity, Consisting of a DNA sequence of SEQ ID NO: 1 of positions 76 to 1515 or a SEQ ID NO: 2 of positions 76 to 1572 or a sequence homology of DNA having at least 80% of the DNA sequence DNA array. An expression vector consisting of the DNA array of claim 1. The method of claim 2, Promoters for regulating the expression of laccase genes in host organisms, and signals for transcription termination, suitable for the host organism and functionally binding to the 3 ′ end of the DNA sequence of claim 1. Expression vector characterized in that it further comprises. The method of claim 3, wherein The promoter is an expression vector characterized in that it is selected from the group of tac promoters, subtilisin promoters, GAL promoters, TAKA amylase promoters, polyhedrin promoters, glucoamylase promoters, GAPDH promoters and alcohol oxidase promoters. . The method according to claim 3 or 4, Expression vector characterized in that it further comprises an N-terminal signal sequence. The method of claim 5, The N-terminal signal sequence is a natural singal sequence present in the laccase gene or a group of signal sequences of the following secreted proteins, and alpha- in Klebsiella oxytoca. Cyclodextrin glucosyltransferase, subtilisin in Bacillus subtilis, alpha-factor in Saccharomyces cerevisiae, pica pastoris group of acid phosphatase in pichia pastoris, alpha-amylase in Aspergillus niger or glucoamylase in Aspergillus niger or Aspergillus awamori Expression vector characterized in that selected from. The method of claim 6, An expression vector characterized by functionally correlating a gene section of a secreted fragment of a gene or a secreted protein gene to a laccase enzyme. Microbial strain consisting of the expression vector of claim 2. Protein sequence consisting of the SEQ ID NO: 3. Clause 9 used for deligination of pulp, depolymerization of polymer aggregates, deinking waste paper, polymerization of organic compounds in lignin-containing wastewater obtained from pulp bleaching as wastewater, oxidation of dyes or activation of dyes to form pigments. A method in which a protein is used for organic synthesis of a coupling reaction of an aromatic compound or a side chain oxidation reaction of an aromatic compound.