LACCASE MUTANTS

Abstract: The present invention relates to laccase mutants with increased oxidation potential and/or changed pH optimum and/or altered mediator pathway and/or altered O2/OH<->pathway.

Description: LACCASE MUTANTS FIELD OF THE INVENTION The present invention relates to laccase mutants with increased oxidation potential and/or changed pH optimum and/or altered mediator pathway and/or altered 0 2 /OH ~ pathway. BACKGROUND OF THE INVENTION Laccase is a polyphenol oxidase (EC 1.10.3.2) which catalyses the oxidation of a variety of inorganic and aromatic compounds, particularly phenols, with the concomitant reduction of molecular oxygen to water. Laccase belongs to a family of blue copper-containing oxidases which includes ascorbate oxidase and the mammalian plasma protein ceruloplasmin. All these enzymes are multi-copper- containing proteins. Because laccases are able to catalyze the oxidation of a variety of inorganic and aromatic compounds, laccases have been suggested in many potential industrial applications such as lignin modification, paper strengthening, dye transfer inhibition in detergents, phenol polymerization, hair colouring, and waste water treatment . The various applications ask for laccases with specific properties. It is the purpose of the present application to create laccase variants with increased oxidation potential and/or changed pH optimum and/or altered mediator pathway and/or altered 0 2 /OH-pathway. BRIEF DISCLOSURE OF THE INVENTION The present invention relates to laccase variants, in particular to - A variant of a parent laccase, which variant has laccase activity, and increased oxidation potential and comprises a mutation in a position corresponding to at least one of the following positions: G511A,V,P,L,I,F,Y,W; T428A,V,P,L,I,F,Y, ; S510A,V,P,L,I,F,Y,W; D106A,V,P,L,I,F,Y, ; N109A,V,P,L,I,F,Y,W,Q; L500I,F,Y,W; A108V,P,L,I,F,Y,W; G514A,V,P,L,I,F,Y,W; wherein the parent laccase has the amino acid sequence given in SEQ ID No. 1 or the parent laccase has an amino acid sequence which is at least 80% homologous to SEQ ID No. 1; - A variant of a parent laccase, which variant has laccase activity and an altered pH optimum and comprises a mutation in a position corresponding to at least one of the following positions : 192-193; 234-236; 269; 293-294 364-365, 372-373 426-433 503-513 wherein the parent laccase has the amino acid sequence given in SEQ ID No. 1 or the parent laccase has an amino acid sequence which is at least 80% homologous to SEQ ID No. 1; - A variant of a parent laccase, which variant has laccase activity and an altered mediator efficiency and comprises a mutation in a position corresponding to at least one of the following positions: 185 - 194 ; 235 ; 293 - 294 , 365 - 373 427 -429 507-508; 510-511; wherein the parent laccase has the amino acid sequence given in SEQ ID No. 1 or the parent laccase has an amino acid sequence which is at least 80% homologous to SEQ ID No. 1; and - A variant of a parent laccase, which variant has laccase activity and an altered 0 2 /OH -pathway and comprises a mutation in a position corresponding to at least one of the following positions : A506E; N109D; H93E; H95E; M433E; M480E; wherein the parent laccase has the amino acid sequence given in SEQ ID No. 1 or the parent laccase has an amino acid sequence which is at least 80% homologous to SEQ ID No. 1. In still further aspects the invention relates to DNA encoding such variants and methods of preparing the variants . Finally, the invention relates to the use of the variants for various industrial purposes . DETAILED DISCLOSURE OF THE INVENTION Homologous Laccases A number of laccases produced by different fungi are homologous on the amino acid level. For instance, when using the homology percent obtained from U GCG program using the GAP program with the default parameters (penalties: gap weight=3.0, length weight=0.1; WISCONSIN PACKAGE Version 8.1-UNIX, August 1995, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) the following homology was found: Myceliophthora thermophila laccase comprising the amino acid sequence shown in SEQ ID No . 1: 100%; Scytalidium thermophilum laccase comprising the amino acid sequence shown in SEQ ID No. 2: 81.2%. Because of the homology found between the above mentioned laccases, they are considered to belong to the same class of laccases, namely the class of "Myceliophthora-like laccases". Accordingly, in the present context, the term " Myceliophthora-like laccase" is intended to indicate a laccase which, on the amino acid level, displays a homology of at least 80% to the Myceliophthora laccase SEQ ID NO 1, or a laccase which, on the amino acid level, displays a homology of at least 85% to the Myceliophthora laccase SEQ ID NO 1, or a laccase which, on the amino acid level, displays a homology of at least 90% to the Myceliophthora laccase SEQ ID NO 1, or a laccase which, on the amino acid level, displays a homology of at least 95% to the Myceliophthora laccase SEQ ID NO 1, or a laccase which, on the amino acid level, displays a homology of at least 98% to the Myceliophthora laccase SEQ ID NO 1. In the present context, "derived from" is intended not only to indicate a laccase produced or producible by a strain of the organism in question, but also a laccase encoded by a DNA sequence isolated from such strain and produced in a host or- ganism containing said DNA sequence. Finally, the term is intended to indicate a laccase which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the laccase in question. Variants with altered oxidation potential The redox potentials of various wild type laccases have been found to be the following (measured at pH 5.3) : E°, V vs NHE Myceliophthora thermophila (SEQ ID No. 1): 0.48 Scytalidium thermophilum (SEQ ID No. 2): 0.53 It is contemplated that it is possible to increase the oxidation potential of a parent laccase, wherein said variant is the result of a mutation, i.e. one or more amino acid residues have been deleted from, replaced or added to the parent laccase. Preferred positions for mutations are the following: Myceliophthora thermophila laccase (SEQ ID No. 1) : G511A,V,P,L,I,F,Y,W; T428A,V,P,L, I,F,Y,W; S510A,V,P,L, I,F,Y,W; D106A,V,P,L, I,F,Y,W; N109A,V,P,L, I,F,Y,W,Q; L500I,F,Y,W; A108V,P,L, I,F,Y,W; G514A,V,P,L, I,F,Y,W; in particular G511A,V,L,I,F; T428V; S510V; D106L; N109I,F,Q; L500F; A108V,I; G514A,V,L,I,F. Preferred variants include any combination of the above mentioned mutations. Variants with altered pH optimum The desired pH optimum of a laccase depends on which application is of interest, e.g., if the laccase is to be used for denim bleaching the preferred pH optimum will be around pH 5- 8, whereas if the laccase is to be used for washing purposes the preferred pH optimum will be around pH 8-10. It is contemplated that it is possible to alter the pH optimum of a parent laccase wherein said variant is the result of a mutation, i.e. one or more amino acid residues have been deleted from, replaced or added to the parent laccase. Preferred positions for mutations are the following: Myceliophthora thermophila (SEQ ID No. 1) : 192-193; 234-236; 269; 293-294, 364-365 372 -373 426 -433 503 - 513 . Preferred substitutions are the following: E, D, L, I, F, Y, W. Variants with altered mediator efficiency Laccases are often used in combination with so called mediators or enhancers, e.g., in combination with phenothiazine or phenothiazine related compounds (see WO 95/01426) or in combination with acetosyringone or acetosyringone related compounds (see WO 96/10079) . It is contemplated that it is possible to alter the mediator efficiency (in order to make the mediator more efficient) , of a parent laccase wherein said variant is the result of a mutation, i.e. one or more amino acid residues have been deleted from, replaced or added to the parent laccase. Preferred positions for mutations are the following: Myceliophthora thermophila laccase (SEQ ID No. 1) : 185-194; 235; 293-294, 365-373 427-429, 505; 507-508; 510-511. Preferred substitutions are in particular one or more of the following mutations: N189G,A,S,T; S190G,A; F371* (deletion) ; F371G,A. Variants with altered 0 /OH-pathwav It is contemplated that it is possible to lower the possibility of OH " entering the trinuclear Cu site by producing one or more of the following mutations: Myceliophthora thermophila (SEQ ID No. 1) : A506E; N109D; H93E; H95E; M433E; M480E. Methods of preparing laccase variants Several methods for introducing mutations into genes are known in the art . After a brief discussion of the cloning of laccase-encoding DNA sequences, methods for generating mutations at specific sites within the laccase-encoding sequence will be discussed. Cloning a DNA sequence encoding a laccase The DNA sequence encoding a parent laccase may be isolated from any cell or microorganism producing the laccase in question, using various methods well known in the art. First, a genomic DNA and/or cDNA library should be constructed using chromosomal DNA or messenger RNA from the organism that produces the laccase to be studied. Then, if the amino acid sequence of the laccase is known, homologous, labelled oligonucleotide probes may be synthesized and used to identify laccase-encoding clones from a genomic library prepared from the organism in question. Alternatively, a labelled oligonucleotide probe containing sequences homologous to a known laccase gene could be used as a probe to identify laccase-encoding clones, using hybridization and washing conditions of lower stringency. A method for identifying laccase-encoding clones involves inserting cDNA into an expression vector, such as a plasmid, transforming laccase-negative fungi with the resulting cDNA library, and then plating the transformed fungi onto agar containing a substrate for laccase, thereby allowing clones expressing the laccase to be identified. Alternatively, the DNA sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g. the phosphoroamidite method. In the phosphoroamidite method, oligonu- cleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors. Finally, the DNA sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate, the fragments corresponding to various parts of the entire DNA sequence) , in accordance with standard techniques . The DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers. Site-directed mutagenesis Once a laccase-encoding DNA sequence has been isolated, and desirable sites for mutation identified, mutations may be introduced using synthetic oligonucleotides . These oligonucleotides contain nucleotide sequences flanking the desired mutation sites; mutant nucleotides are inserted during oligonucleotide synthesis. In a specific method, a single-stranded gap of DNA, bridging the laccase-encoding sequence, is created in a vector carrying the laccase gene. Then the synthetic nucleotide, bearing the desired mutation, is annealed to a homologous portion of the single- stranded DNA. The remaining gap is then filled in with T7 DNA polymerase and the construct is ligated using T4 ligase. A specific example of this method is described in Morinaga et al . (1984). US 4,760,025 discloses the introduction of oligonucleoti- des encoding multiple mutations by performing minor alterations of the cassette. However, an even greater variety of mutations can be introduced at any one time by the Morinaga method, because a multitude of oligonucleotides, of various lengths, can be introduced. Another method of introducing mutations into laccase-encoding DNA sequences is described in Nelson and Long (1989) . It involves the 3 -step generation of a PCR fragment containing the desired mutation introduced by using a chemically synthesized DNA strand as one of the primers in the PCR reactions. From the PCR-gener- ated fragment, a DNA fragment carrying the mutation may be isolated by cleavage with restriction endonucleases and reinserted into an expression plasmid. Random mutagenesis The random mutagenesis of a DNA sequence encoding a parent laccase may conveniently be performed by use of any method known in the art . For instance, the random mutagenesis may be performed by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the random mutagenesis may be performed by use of any combination of these mutagenizing agents . The mutagenizing agent may, e.g., be one which induces transitions, transversions, inversions, scrambling, deletions, and/or insertions . Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N' -nitro-N-nitrosoguanidine (MNNG) , O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS) , sodium bisulphite, formic acid, and nucleotide analogues . When such agents are used, the mutagenesis is typically performed by incubating the DNA sequence encoding the parent enzyme to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions for the mutagenesis to take place, and selecting for mutated DNA having the desired properties . When the mutagenesis is performed by the use of an oligonucleotide, the oligonucleotide may be doped or spiked with the three non-parent nucleotides during the synthesis of the oligonucleotide at the positions which are to be changed. The doping or spiking may be done so that codons for unwanted amino acids are avoided. The doped or spiked oligonucleotide can be incorporated into the DNA encoding the laccase enzyme by any published technique, using e.g. PCR, LCR or any DNA polymerase and ligase . When PCR-generated mutagenesis is used, either a chemically treated or non-treated gene encoding a parent laccase enzyme is subjected to PCR under conditions that increase the mis- incorporation of nucleotides (Deshler 1992; Leung et al . , Technique, Vol.l, 1989, pp. 11-15). A mutator strain of E. coli (Fowler et al . , Molec . Gen. Genet., 133, 1974, pp. 179-191), S. cereviseae or any other microbial organism may be used for the random mutagenesis of the DNA encoding the laccase enzyme by e.g. transforming a plasmid containing the parent enzyme into the mutator strain, growing the mutator strain with the plasmid and isolating the mutated plasmid from the mutator strain. The mutated plasmid may subsequently be transformed into the expression organism. The DNA sequence to be mutagenized may conveniently be present in a genomic or cDNA library prepared from an organism expressing the parent laccase enzyme. Alternatively, the DNA se- quence may be present on a suitable vector such as a plasmid or a bacteriophage, which as such may be incubated with or otherwise exposed to the mutagenizing agent. The DNA to be mutagenized may also be present in a host cell either by being integrated in the genome of said cell or by being present on a vector harboured in the cell. Finally, the DNA to be mutagenized may be in isolated form. It will be understood that the DNA sequence to be subjected to random mutagenesis is preferably a cDNA or a genomic DNA sequence . In some cases it may be convenient to amplify the mutated DNA sequence prior to the expression step or the screening step being performed. Such amplification may be performed in accordance with methods known in the art, the presently preferred method being PCR-generated amplification using oligonucleotide primers prepared on the basis of the DNA or amino acid sequence of the parent enzyme . Subsequent to the incubation with or exposure to the mutagenizing agent, the mutated DNA is expressed by culturing a suitable host cell carrying the DNA sequence under conditions allowing expression to take place. The host cell used for this purpose may be one which has been transformed with the mutated DNA sequence, optionally present on a vector, or one which was carried the DNA sequence encoding the parent enzyme during the mutagenesis treatment. Examples of suitable host cells are fungal hosts such as Aspergillus niger or Aspergillus oryzae . The mutated DNA sequence may further comprise a DNA sequence encoding functions permitting expression of the mutated DNA sequence . Localized random mutagenesis The random mutagenesis may advantageously be localized to a part of the parent laccase in question. This may, e.g., be advantageous when certain regions of the enzyme have been identified to be of particular importance for a given property of the enzyme, and when modified are expected to result in a variant having improved properties. Such regions may normally be identified when the tertiary structure of the parent enzyme has been elucidated and related to the function of the enzyme. The localized random mutagenesis is conveniently performed by use of PCR-generated mutagenesis techniques as described above or any other suitable technique known in the art . Alternatively, the DNA sequence encoding the part of the DNA sequence to be modified may be isolated, e.g. by being inserted into a suitable vector, and said part may subsequently be subjected to mutagenesis by use of any of the mutagenesis methods discussed above. With respect to the screening step in the above-mentioned method of the invention, this may conveniently be performed by use of aa filter assay based on the following principle: A microorganism capable of expressing the mutated laccase enzyme of interest is incubated on a suitable medium and under suitable conditions for the enzyme to be secreted, the medium being provided with a double filter comprising a first protein- binding filter and on top of that a second filter exhibiting a low protein binding capability. The microorganism is located on the second filter. Subsequent to the incubation, the first filter comprising enzymes secreted from the microorganisms is separated from the second filter comprising the microorganisms. The first filter is subjected to screening for the desired enzymatic activity and the corresponding microbial colonies present on the second filter are identified. The filter used for binding the enzymatic activity may be any protein binding filter e.g. nylon or nitrocellulose. The top filter carrying the colonies of the expression organism may be any filter that has no or low affinity for binding proteins e.g. cellulose acetate or Durapore™. The filter may be pretreated with any of the conditions to be used for screening or may be treated during the detection of enzymatic activity. The enzymatic activity may be detected by a dye, fluorescence, precipitation, pH indicator, IR-absorbance or any other known technique for detection of enzymatic activity. The detecting compound may be immobilized by any immobilizing agent, e.g., agarose, agar, gelatine, polyacrylamide, starch, filter paper, cloth; or any combination of immobilizing agents. Laccase activity In the context of this invention, the laccase activity was measured using 10- (2-hydroxyethyl) -phenoxazine (HEPO) as substrate for the various laccases. HEPO was synthesized using the same procedure as described for 10- (2-hydroxyethyl) - phenothiazine, (G. Cauquil in Bulletin de la Society Chemique de France, 1960, p. 1049) . In the presence of oxygen laccases (E.C. 1.10.3.2) oxidize HEPO to a HEPO radical that can be monitored photometrically at 528 nm. The Myceliophthora thermophila laccase was measured using 0.4 mM HEPO in 25 mM Tris-HCl, pH 7.5, 0.05% TWEEN-20 at 30 °C. The absorbance at 528 nm was followed for 200 s and the rate calculated from the linear part of the progress curve. Expression of laccase variants According to the invention, a DNA sequence encoding the variant produced by methods described above, or by any alterna- tive methods known in the art, can be expressed, in enzyme form, using an expression vector which typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes. The recombinant expression vector carrying the DNA sequence encoding a laccase variant of the invention may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid, a bacteriophage or an extrachromosomal element, minichromosome or an artificial chromosome. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome (s) into which it has been integrated. In the vector, the DNA sequence should be operably connected to a suitable promoter sequence . The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA sequence encoding a laccase variant of the invention, especially in a fungal host, are those derived from the gene encoding A . oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A . niger neutral α-amylase, A . niger acid stable α-amylase, A . niger glu- coamylase, Rhizomucor miehei lipase, A . oryzae alkaline protease, A. oryzae triose phosphate isomerase or A . nidulans acetamidase . The expression vector of the invention may also comprise a suitable transcription terminator and, in eukaryotes, poly- adenylation sequences operably connected to the DNA sequence encoding the laccase variant of the invention. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter. The vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUBHO, pE194, pAMBl and pIJ702. The vector may also comprise a selectable marker, e.g. a gene, the product of which complements a defect in the host cell, such as one which confers antibiotic resistance such as ampicil- lin, kanamycin, chloramphenicol or tetracyclin resistance. Furthermore, the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and sC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, e.g. as described in WO 91/17243. The procedures used to ligate the DNA construct of the inven- tion encoding a laccase variant, the promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al . (1989)). The cell of the invention, either comprising a DNA construct or an expression vector of the invention as defined above, is advantageously used as a host cell in the recombinant production of a laccase variant of the invention. The cell may be transformed with the DNA construct of the invention encoding the variant, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g. by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells. The cell of the invention may be a cell of a higher organism such as a mammal or an insect, but is preferably a microbial cell, e.g. a fungal cell. The filamentous fungus may advantageously belong to a species of Aspergillus, e.g. Aspergillus oryzae or Aspergillus niger. Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se . A suitable procedure for transformation of Aspergillus host cells is described in EP 238 023. In a yet further aspect, the present invention relates to a method of producing a laccase variant of the invention, which method comprises cultivating a host cell as described above under conditions conducive to the production of the variant and recovering the variant from the cells and/or culture medium. The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of the laccase variant of the invention. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. as described in catalogues of the American Type Culture Collection) . The laccase variant secreted from the host cells may con- veniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like. Industrial Applications The laccase variants of this invention possesses valuable properties allowing for various industrial applications, in particular lignin modification, paper strengthening, dye transfer inhibition in detergents, phenol polymerization, hair dyeing, textile dyeing, bleaching of textiles (in particular bleaching of denim as described in WO 96/12845 and WO 96/12846) and waste water treatment . SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: NOVO NORDISK A/S (B) STREET: Novo Alle (C) CITY: Bagsvaerd (E) COUNTRY: Denmark (F) POSTAL CODE (ZIP) : DK-2880 (G) TELEPHONE: +45 44 44 88 88 (H) TELEFAX: +45 44 49 05 55 (ii) TITLE OF INVENTION: LACCASE MUTANTS (iii) NUMBER OF SEQUENCES: 2 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO : 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 573 amino acids (B) TYPE: amino acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Gin Gin Ser Cys Asn Thr Pro Ser Asn Arg Ala Cys Trp Thr Asp Gly 1 5 10 15 Tyr Asp He Asn Thr Asp Tyr Glu Val Asp Ser Pro Asp Thr Gly Val Val Arg Pro Tyr Thr Leu Thr Leu Thr Glu Val Asp Asn Trp Thr Gly 35 40 45 Pro Asp Gly Val Val Lys Glu Lys Val Met Leu Val Asn Asn Ser He 50 55 60 He Gly Pro Thr He Phe Ala Asp Trp Gly Asp Thr He Gin Val Thr 65 70 75 80 Val He Asn Asn Leu Glu Thr Asn Gly Thr Ser He His Trp His Gly 85 90 95 Leu His Gin Lys Gly Thr Asn Leu His Asp Gly Ala Asn Gly He Thr 100 105 110 Glu Cys Pro He Pro Pro Lys Gly Gly Arg Lys Val Tyr Arg Phe Lys 115 120 125 Ala Gin Gin Tyr Gly Thr Ser Trp Tyr His Ser His Phe Ser Ala Gin 130 135 140 Tyr Gly Asn Gly Val Val Gly Ala He Gin He Asn Gly Pro Ala Ser 145 150 155 160 Leu Pro Tyr Asp Thr Asp Leu Gly Val Phe Pro He Ser Asp Tyr Tyr 165 170 175 Tyr Ser Ser Ala Asp Glu Leu Val Glu Leu Thr Lys Asn Ser Gly Ala 180 185 190 Pro Phe Ser Asp Asn Val Leu Phe Asn Gly Thr Ala Lys His Pro Glu 195 200 205 Thr Gly Glu Gly Glu Tyr Ala Asn Val Thr Leu Thr Pro Gly Arg Arg 210 215 220 His Arg Leu Arg Leu He Asn Thr Ser Val Glu Asn His Phe Gin Val 225 230 235 240 Ser Leu Val Asn His Thr Met Cys He He Ala Ala Asp Met Val Pro 245 250 255 Val Asn Ala Met Thr Val Asp Ser Leu Phe Leu Gly Val Gly Gin Arg 260 265 270 Tyr Asp Val Val He Glu Ala Asn Arg Thr Pro Gly Asn Tyr Trp Phe 275 280 285 Asn Val Thr Phe Gly Gly Gly Leu Leu Cys Gly Gly Ser Arg Asn Pro 290 295 300 Tyr Pro Ala Ala He Phe His Tyr Ala Gly Ala Pro Gly Gly Pro Pro 305 310 315 320 Thr Asp Glu Gly Lys Ala Pro Val Asp His Asn Cys Leu Asp Leu Pro 325 330 335 Asn Leu Lys Pro Val Val Ala Arg Asp Val Pro Leu Ser Gly Phe Ala 340 345 350 Lys Arg Ala Asp Asn Thr Leu Asp Val Thr Leu Asp Thr Thr Gly Thr 355 360 365 Pro Leu Phe Val Trp Lys Val Asn Gly Ser Ala He Asn He Asp Trp 370 375 380 Gly Arg Ala Val Val Asp Tyr Val Leu Thr Gin Asn Thr Ser Phe Pro 385 390 395 400 Pro Gly Tyr Asn He Val Glu Val Asn Gly Ala Asp Gin Trp Ser Tyr 405 410 415 Trp Leu He Glu Asn Asp Pro Gly Ala Pro Phe Thr Leu Pro His Pro 420 425 430 Met His Leu His Gly His Asp Phe Tyr Val Leu Gly Arg Ser Pro Asp 435 440 445 Glu Ser Pro Ala Ser Asn Glu Arg His Val Phe Asp Pro Ala Arg Asp 450 455 460 Ala Gly Leu Leu Ser Gly Ala Asn Pro Val Arg Arg Asp Val Ser Met 465 470 475 480 Leu Pro Ala Phe Gly Trp Val Val Leu Ser Phe Arg Ala Asp Asn Pro 485 490 495 Gly Ala Trp Leu Phe His Cys His He Ala Trp His Val Ser Gly Gly 500 505 510 Leu Gly Val Val Tyr Leu Glu Arg Ala Asp Asp Leu Arg Gly Ala Val 515 520 525 Ser Asp Ala Asp Ala Asp Asp Leu Asp Arg Leu Cys Ala Asp Trp Arg 530 535 540 Arg Tyr Trp Pro Thr Asn Pro Tyr Pro Lys Ser Asp Ser Gly Leu Lys 545 550 555 560 His Arg Trp Val Glu Glu Gly Glu Trp Leu Val Lys Ala 565 570 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 616 amino acids (B) TYPE: amino acid (C) STRANDEDNESS : (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2: Met Lys Arg Phe Phe He Asn Ser Leu Leu Leu Leu Ala Gly Leu Leu 1 5 10 15 Asn Ser Gly Ala Leu Ala Ala Pro Ser Thr His Pro Arg Ser Asn Pro 20 25 30 Asp He Leu Leu Glu Arg Asp Asp His Ser Leu Thr Ser Arg Gin Gly 35 40 45 Ser Cys His Ser Pro Ser Asn Arg Ala Cys Trp Cys Ser Gly Phe Asp 50 55 60 He Asn Thr Asp Tyr Glu Thr Lys Thr Pro Asn Thr Gly Val Val Arg 65 70 75 80 Arg Tyr Thr Phe Asp He Thr Glu Val Asp Asn Arg Pro Gly Pro Asp 85 90 95 Gly Val He Lys Glu Lys Leu Met Leu He Asn Asp Lys Leu Leu Gly 100 105 110 Pro Thr Val Phe Ala Asn Trp Gly Asp Thr He Glu Val Thr Val Asn 115 120 125 Asn His Leu Arg Thr Asn Gly Thr Ser He His Trp His Gly Leu His 130 135 140 Gin Lys Gly Thr Asn Tyr His Asp Gly Ala Asn Gly Val Thr Glu Cys 145 150 155 160 Pro He Pro Pro Gly Gly Ser Arg Val Tyr Ser Phe Arg Ala Arg Gin 165 170 175 Tyr Gly Thr Ser Trp Tyr His Ser His Phe Ser Ala Gin Tyr Gly Asn 180 185 190 Gly Val Ser Gly Ala He Gin He Asn Gly Pro Ala Ser Leu Pro Tyr 195 200 205 Asp He Asp Leu Gly Val Leu Pro Leu Xaa Asp Trp Tyr Tyr Lys Ser 210 215 220 Ala Asp Gin Leu Val He Glu Thr Leu Xaa Lys Gly Asn Ala Pro Phe 225 230 235 240 Ser Asp Asn Val Leu He Asn Gly Thr Ala Lys His Pro Thr Thr Gly 245 250 255 Glu Gly Glu Tyr Ala He Val Lys Leu Thr Pro Asp Lys Arg His Arg 260 265 270 Leu Arg Leu He Asn Met Ser Val Glu Asn His Phe Gin Val Ser Leu 275 280 285 Ala Lys His Thr Met Thr Val He Ala Ala Asp Met Val Pro Val Asn 290 295 300 Ala Met Thr Val Asp Ser Leu Phe Met Ala Val Gly Gin Arg Tyr Asp 305 310 315 320 Val Thr He Asp Ala Ser Gin Ala Val Gly Asn Tyr Trp Phe Asn He 325 330 335 Thr Phe Gly Gly Gin Gin Lys Cys Gly Phe Ser His Asn Pro Ala Pro 340 345 350 Ala Ala He Phe Arg Tyr Glu Gly Ala Pro Asp Ala Leu Pro Thr Asp 355 360 365 Pro Gly Ala Ala Pro Lys Asp His Gin Cys Leu Asp Thr Leu Asp Leu 370 375 380 Ser Pro Val Val Gin Lys Asn Val Pro Val Asp Gly Phe Val Lys Glu 385 390 395 400 Pro Gly Asn Thr Leu Pro Val Thr Leu His Val Asp Gin Ala Ala Ala 405 410 415 Pro His Val Phe Thr Trp Lys He Asn Gly Ser Ala Ala Asp Val Asp 420 425 430 Trp Asp Arg Pro Val Leu Glu Tyr Val Met Asn Asn Asp Leu Ser Ser 435 440 445 He Pro Val Lys Asn Asn He Val Arg Val Asp Gly Val Asn Glu Trp 450 455 460 Thr Tyr Trp Leu Val Glu Asn Asp Pro Glu Gly Arg Leu Ser Leu Pro 465 470 475 480 His Pro Met His Leu His Gly His Asp Phe Phe Val Leu Gly Arg Ser 485 490 495 Pro Asp Val Ser Pro Asp Ser Glu Thr Arg Phe Val Phe Asp Pro Ala 500 505 510 Val Asp Leu Pro Arg Leu Arg Gly His Asn Pro Val Arg Arg Asp Val 515 520 525 Thr Met Leu Pro Ala Arg Gly Trp Leu Leu Leu Ala Phe Arg Thr Asp 530 535 540 Asn Pro Gly Ala Trp Leu Phe His Cys His He Ala Xaa His Val Ser 545 550 555 560 Gly Gly Leu Ser Val Asp Phe Leu Glu Arg Pro Asp Glu Leu Arg Gly 565 570 575 Gin Leu Thr Gly Glu Ser Lys Ala Glu Leu Glu Arg Val Cys Arg Glu 580 585 590 Trp Lys Asp Trp Glu Ala Lys Ser Pro His Gly Lys He Asp Ser Gly 595 600 605 Leu Lys Gin Arg Arg Trp Asp Ala 610 615

Claims: CLAIMS 1. A variant of a parent laccase, which variant has laccase activity, and increased oxidation potential and comprises a 5 mutation in a position corresponding to at least one of the following positions: G511A,V,P,L,I,F,Y,W; T428A,V,P,L,I,F,Y,W; S510A,V,P,L,I,F,Y,W; 10 D106A,V,P,L,I,F,Y,W; N109A,V,P,L,I,F,Y,W,Q; L500I,F,Y,W; A108V,P,L,I,F,Y,W; G514A,V,P,L, I,F,Y,W; 15 wherein the parent laccase has the amino acid sequence given in SEQ ID No. 1 or the parent laccase has an amino acid sequence which is at least 80% homologous to SEQ ID No. 1. 2. A variant of a parent laccase, which variant has laccase 20 activity and an altered pH optimum and comprises a mutation in a position corresponding to at least one of the following positions : 192-193; 234-236; 25269; 293-294 364-365, 372-373 426-433 30503-513 wherein the parent laccase has the amino acid sequence given in SEQ ID No . 1 or the parent laccase has an amino acid sequence which is at least 80% homologous to SEQ ID No . 1. 353. A variant of a parent laccase, which variant has laccase activity and an altered mediator efficiency and comprises a mutation in a position corresponding to at least one of the following positions: 185-194; 235; 293-294 5365-373 427-429, 505; 507-508; 510-511; 0 wherein the parent laccase has the amino acid sequence given in SEQ ID No. 1 or the parent laccase has an amino acid sequence which is at least 80% homologous to SEQ ID No. 1. 4. A variant of a parent laccase, which variant has laccase 5 activity and an altered 0 2 /OH " -pathway and comprises a mutation in a position corresponding to at least one of the following positions : A506E; N109D; 0 H93E; H95E; M433E; M480E; wherein the parent laccase has the amino acid sequence given in 5 SEQ ID No . 1 or the parent laccase has an amino acid sequence which is at least 80% homologous to SEQ ID No. 1. 5. A variant according to any of claims 1-4, wherein the parent laccase is derived from Myceliophthora . 0 6. A variant according to any of claims 1-4, wherein the parent laccase is derived from Scytalidium . 7. A variant according to claim 5, wherein the parent laccase 5 is a Scytalidium thermophilum laccase with the sequence ID No. 2. 8. A DNA construct comprising a DNA sequence encoding a laccase variant according to any of claims 1-7. 9. A recombinant expression vector which carries a DNA con- 5 struct according to claim 8. 10. A cell which is transformed with a DNA construct according to claim 8 or a vector according to claim 9. 1011. A cell according to claim 10, which is a microorganism. 12. A cell according to claim 11, which is a bacterium or a fungus . 1513. A cell according to claim 12, which is an Aspergillus niger or an Aspergillus oryzae cell. 14. Use of a laccase variant according to any of claims 1-7 for oxidizing a substrate. 20 15. Use of a laccase variant according to any of claims 1-7 for dye transfer inhibition. 16. Use of a laccase variant according to any of claims 1-7 for 25 bleaching textiles, in particular for bleaching denim. 17. A detergent additive comprising a laccase variant according to any of claims 1-7 in the form of a non-dusting granulate, a stabilised liquid or a protected enzyme. 30 18. A detergent additive according to claim 17, which additionally comprises one or more other enzyme such as a protease, a lipase, an amylase, and/or a cellulase. 3519. A detergent composition comprising a laccase variant according to any of claims 1-7 and a surfactant. 20. A detergent composition according to claim 19 which additionally comprises one or more other enzymes such as a protease, a lipase, an amylase and/or a cellulase.

Inventors: SVENDSEN ALLAN, SVENDSEN, ALLAN
Applicants: NOVONORDISK AS, NOVO NORDISK A/S
Application year: 1998
Main classification: C12N9
IPC codes: C12N9