WO1996000290A1 - Purified polyporus laccases and nucleic acids encoding same - Google Patents

Abstract

The present invention relates to isolated nucleic acid constructs containing a sequence encoding a Polyporus laccase, and the laccase proteins encoded thereby.

Description

PURIFIED POLYPORUS LACCASES AND NUCLEIC ACIDS

ENCODING SAME

Field of the Invention

The present invention relates to isolated nucleic acid fragments encoding a fungal oxidoreductase enzyme and the purified enzymes produced thereby. More particularly, the invention relates to nucleic acid fragments encoding a phenol oxidase, specifically a laccase, of a basidiomycete, Polyporus. Background of the Invention

Laccases (benzenediol: oxygen oxidoreductases) are multi-copper-containing enzymes that catalyze the oxidation of phenolics. Laccase-mediated oxidations result in the production of aryloxy-radical intermediates from suitable phenolic substrate; the ultimate coupling of the

intermediates so produced provides a combination of dimeric, oligomeric, and polymeric reaction products. Such reactions are important in nature in biosynthetic pathways which lead to the formation of melanin, alkaloids, toxins, lignins, and humic acids. Laccases are produced by a wide variety of fungi, including ascomycetes such as Aspergillus,

Neurospora, and Podospora, the deuteromycete Botrytis, and basidiomycetes such as Collybia, Fomes, Lentinus, PIeurotus, Trametes, Polyporus and perfect forms of Rhizoctonia.

Laccases exhibit a wide range of substrate specificity, and each different fungal laccase usually differs only

quantitatively from others in its ability to oxidize

phenolic substrates. Because of the substrate diversity, laccases generally have found many potential industrial applications. Among these are lignin modification, paper strengthening, dye transfer inhibition in detergents, phenol polymerization, juice manufacture, phenol resin production, and waste water treatment.

Although the catalytic capabilities are similar, laccases made by different fungal species do have different temperature and pH optima, and these may also differ depending on the specific substrate. A number of these fungal laccases have been isolated, and the genes for several of these have been cloned. For example, Choi et al . (Mol. Plant-Microbe Interactions 5: 119-128, 1992) describe the molecular characterization and cloning of the gene encoding the laccase of the chestnut blight fungus, Cryphonectria parasitica . Kojima et al . (J. Biol. Chem. 265: 15224-15230, 1990; JP 2-238885) provide a description of two allelic forms of the laccase of the white-rot basidiomycete Coriolus hirsutus . Germann and Lerch

(Experientia 41: 801,1985; PNAS USA 83: 8854-8858, 1986) have reported the cloning and partial sequencing of the Neurospora crassa laccase gene. Saloheimo et al . (J. Gen. Microbiol. 137: 1537-1544, 1985; WO 92/01046) have

disclosed a structural analysis of the laccase gene from the fungus Phlebia radiata.

Attempts to express laccase genes in heterologous fungal systems frequently give very low yields (Kojima et al., supra; Saloheimo et al., Bio/Technol. 9 : 987-990, 1991). For example, heterologous expression of Phlebia radiata laccase in Trichoderma reesei gave only 20 mg per liter of active enzyme in lab-scale fermentation (Saloheimo, 1991, supra). Although laccases have great commercial potential, the ability to express the enzyme in significant quantities is critical to their commercial utility.

Previous attempts to express basidiomycete laccases in recombinant hosts have resulted in very low yields. The present invention now provides novel basidiomycete laccases which are well expressed in Aspergillus .

Summary of the Invention

The present invention relates to a DNA construct containing a nucleic acid sequence encoding a Polyporus laccase. The invention also relates to an isolated laccase encoded by the nucleic acid sequence. Preferably, the laccase is substantially pure. By "substantially pure" is meant a laccase which is essentially (i.e.,≥90%) free of other non-laccase proteins.

In order to facilitate production of the novel laccase, the invention also provides vectors and host cells

comprising the claimed nucleic acid sequence, which vectors and host cells are useful in recombinant production of the laccase. The sequence is operably linked to transcription and translation signals capable of directing expression of the laccase protein in the host cell of choice. A preferred host cell is a fungal cell, most preferably of the genus Aspergillus . Recombinant production of the laccase of the invention is achieved by culturing a host cell transformed or transfected with the construct of the invention, or progeny thereof, under conditions suitable for expression of the laccase protein, and recovering the laccase protein from the culture.

The laccases of the present invention are useful in a number of industrial processes in which oxidation of

phenolics is required. These processes include lignin manipulation, juice manufacture, phenol polymerization and phenol resin production.

Brief Description of the Figures

Figure 1 shows the DNA sequence and translation of genomic clone 21GEN, containing LCC1 (SEQ ID NO. 1) Figure 2 shows the DNA sequence and translation of genomic clone 23GEN, containing LCC2 (SEQ ID NO. 3)

Figure 3 shows the DNA sequence and translation of genomic clone 24GEN, containing LCC3 (SEQ ID NO. 5)

Figure 4 shows the DNA sequence and translation of genomic clone 31GEN, containing LCC4 (SEQ ID NO. 7)

Figure 5 shows the DNA sequence and translation of genomic clone 41GEN, containing LCC5 (SEQ ID NO. 9)

Figure 6 shows the structure of vector pMWRl

Figure 7 shows the structure of vector pDSYl

Figure 8 shows the structure of vector pDSYlO

Figure 9 shows the pH profile of the laccase produced by pDSY2; (A) syringaldazine oxidation; (B) ABTS oxidation.

Figure 10 illustrates a comparison of the use of laccase vs. H₂O₂, with various dye precursors, in hair dyeing, as a measurement of DL*.

Figure 11 illustrates a comparison of the use of laccase vs. H₂O₂, with various dye precursors, in hair dyeing as a measurement of Da*.

Figure 12 illustrates a comparison of the use of laccase vs. H₂O₂, with various dye precursors and modifiers, in hair dyeing, as a measurement of DL*.

Figure 13 illustrates a comparison of the wash

stability of hair dyed with laccase vs. H₂O₂.

Figure 14 illustrates the light fastness of hair dyed with laccase vs. H₂O₂.

Detailed Description of the Invention

Polyporus pinsitus is a basidiomycete, also referred to as Trametes villosa . Polyporus species have previously been identified as laccase producers (Fahraeus and Lindeberg,

Physiol. Plant. 6 : 150-158, 1953). However, there has been no previous description of a purified laccase from Polyporus pinsitus. It has now been determined that Polyporus pinsitus produces at least two different laccases, and the genes encoding these laccases can be used to produce

relatively large yields of the enzyme in convenient host systems such as Aspergillus . In addition, three other genes which appear to code for laccases have also been isolated.

Initial screenings of a variety of fungal strains indicate that Polyporus pinisitus is a laccase producer. The production of laccase by P. pinsitus is induced by 2,5- xylidine. Attempts are first initiated to isolate the laccase from the supernatant of the induced strains. Anion exchange chromatography identifies an approximately 65 kD(on SDS-PAGE) protein which exhibits laccase activity. The enzyme is purified sufficiently to provide several internal peptide sequences, as well as an N-terminal sequence. The initial sequence information indicates the laccase has significant homology to that of Coriolus hirsutus, as well as to an unidentified basidiomycete laccase (Coll et al., Appl. Environ. Microbiol. 59: 4129-4135, 1993. Based on th sequence information, PCR primers are designed and PCR carried out on cDNA isolated from P. pinsitus . A band of the expected size is obtained by PCR, and the isolated fragment linked to a cellulase signal sequence is shown to express an active laccase in A. oryzae, but at low levels. One of the PCR fragments is also used as a probe in

screening a P. pinsitus cDNA library. In this manner, more than 100 positive clones are identified. The positive clones are characterized and the ends of the longest clones sequenced; none of the clones are found to be full-length.

Further attempts to isolate a full length clone are made. A 5-6 kb BamHI size-selected P. pinsi tus genomic library is probed with the most complete cDNA fragment isolated as described above. Initial screening identifies one clone 24GEN(LCC3) having homology to the cDNA, but which is not the cDNA-encoded laccase and also not full length. Subsequent screening of a 7-8kb BamHI/EcoRi size-selected library indicates the presence of at least two laccases; partial sequencing shows that one, called 21GEN(LCC1), is identical to the original partial cDNA clone isolated, and the second, called 31GEN(LCC4) is a new, previously

unidentified laccase. Secondary screenings of an EMBL4 genomic bank with LCC1 as probe identifies a class of clone containing the entire LCC1 insert as well as the 5' and 3' flanking regions. Screening of the EMBL bank with LCC3 identifies two additional clones encoding laccases which had not previously been identified, 41GEN(LCC5) and 23GEN(LCC2) and which differed structurally from the other three clones LCC1, LCC3, and LCC4. The nucleic acid and predicted amino acid sequences of each of the laccases is presented in

Figures 1-5, and in SEQ ID NOS. 1-10. A comparison of the structural organization of each of the laccases is presented in Table 2. The laccases are generally optimally active at acid pH, between about 4-5.5.

LCC1 is used to create expression vectors, which are in turn used to transform various species of Aspergillus.

Transformation is successful in all species tested, although expression levels are highest in Aspergillus niger. Shake flask cultures are capable of producing 15 or more mg/liter of laccase, and in lab-scale fermentors, yields of over 300mg/liter are observed. This is a significant improvement over laccase levels observed previously with other laccases and other fungal host cells.

According to the invention, a Polyporus gene encoding a laccase can be obtained by methods described above, or any alternative methods known in the art, using the information provided herein. The gene can be expressed, in active form, using an expression vector. A useful expression vector contains an element that permits stable integration of the vector into the host cell genome or autonomous replication of the vector in a host cell independent of the genome of the host cell, and preferably one or more phenotypic markers which permit easy selection of transformed host cells. The expression vector may also include control sequences

encoding a promoter, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes. To permit the secretion of the expressed protein, nucleotides encoding a signal sequence may be inserted prior to the coding sequence of the gene. For expression under the direction of control sequences, a laccase gene to be used according to the invention is operably linked to the control sequences in the proper reading frame. Promoter sequences that can be incorporated into plasmid vectors, and which can direct the transcription of the laccase gene, include but are not limited to the prokaryotic β-lactamase promoter (Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731) and the tac promoter (DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25). Further references can also be found in "Useful proteins from recombinant bacteria" in Scientific American, 1980,, 242:74-94; and in Sambrook et al., Molecular Cloning, 1989.

The expression vector carrying the DNA construct of the invention may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will typically 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, 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 laccase 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 construct of the invention,

especially in a bacterial host, are the promoter of the lac operon of E. coli , the Streptomyces coelicolor agarase gene dagA promoters, the promoters of the Bacillus licheniformis α-amylase gene (amyL), the promoters of the Bacillus

stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens α-amylase

(amyQ), or the promoters of the Bacillus subtilis xylA and xylB genes. In a yeast host, a useful promoter is the eno-1 promoter. For transcription in a fungal host, examples of useful promoters 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 or A . awamori glucoamylase ( glaA), Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase. Preferred are the TAKA-amylase and glaA promoters.

The expression vector of the invention may also

comprise a suitable transcription terminator and, in

eukaryotes, polyadenylation sequences operably connected to the DNA sequence encoding the laccase 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, pUB110, pE194, pAMB1 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 the dal genes from B. subtilis or B. li - cheniformis, or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Examples of Aspergillus selection markers include amdS, pyrG, argB, niaD, sC, trpC and hygB, a marker giving rise to hygromycin resistance. Preferred for use in an Aspergillus host cell are the amdS and pyrG markers of A. nidulans or A. oryzae. A frequently used mammalian marker is the dihydrofolate reductase (DHFR) gene. Furthermore, selection may be accomplished by co-transformation, e.g. as described in WO 91/17243.

It is generally preferred that the expression gives rise to a product which is extracellular. The laccases of the present invention may thus comprise a preregion

permitting secretion of the expressed protein into the culture medium. If desirable, this preregion may be native to the laccase of the invention or substituted with a different preregion or signal sequence, conveniently accomplished by substitution of the DNA sequences encoding the respective preregions. For example, the preregion may be derived from a glucoamylase or an amylase gene from an Aspergillus species, an amylase gene from a Bacillus species, a lipase or proteinase gene from Rhizomucor miehei , the gene for the α-factor from Saccharomyces cerevisiae or the calf

preprochymosin gene. Particularly preferred, when the host is a fungal cell, is the signal sequence for A. oryzae TAKA amylase, A. niger neutral amylase, the Rhizomucor miehei aspartic proteinase signal, the Rhizomucor miehei lipase signal, the maltogenic amylase from Bacillus NCIB 11837, B. stearothermophilus α-amylase, or B. licheniformis

subtilisin.

The procedures used to ligate the DNA construct of the invention, the promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to p.ersons skilled in the art (cf., for instance, Sambrook et al. Molecular Cloning, 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 enzyme of the invention. The cell may be transformed with the DNA construct of the invention, conveniently by integrating the DNA construct 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 host cell may be selected from prokaryotic cells, such as bacterial cells. Examples of suitable bacteria are gram positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus

amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus

thuringiensis, or Streptomyces lividans or Strep tomyces murinus , or gram negative bacteria such as E. coli . The transformation of the bacteria may for instance be effected by protoplast transformation or by using competent cells in a manner known per se.

The host cell may also be a eukaryote, such as

mammalian cells, insect cells, plant cells or preferably fungal cells, including yeast and filamentous fungi. For example, useful mammalian cells include CHO or COS cells. A yeast host cell may be selected from a species of

Saccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae. Useful filamentous fungi may be selected from a species of Aspergillus, e.g. Aspergillus oryzae or

Aspergillus niger. Alternatively, a strain of a Fusarium species, e.g. F. oxysporum, can be used as a host cell.

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. A suitable method of transforming Fusarium species is described by Malardier et al., 1989.

The present invention thus provides a method of

producing a recombinant laccase of the invention, which method comprises cultivating a host cell as described above under conditions conducive to the production of the enzyme and recovering the enzyme 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 of the invention. Suitable media are available from commercial suppliers or may be prepared according to published formulae (e.g. in catalogues of the American Type Culture

Collection). In a preferred embodiment, the recombinant production of laccase in culture is achieved in the presence of an excess amount of copper. Although trace metals added to the culture medium typically contain a small amount of copper, experiments conducted in connection with the present

invention show that addition of a copper supplement to the medium can increase the yield of active enzyme many-fold. Preferably, the copper is added to the medium in soluble form, preferably in the form of a soluble copper salt, such as copper chloride, copper sulfate, or copper acetate. The final concentration of copper in the medium should be in the range of from 0.2-2mM, and preferably in the range of from 0.05-0.5mM. This method can be used in enhancing the yield of any recombinantly produced fungal laccase, as well as other copper-containing enzymes, in particular

oxidoreductases.

The resulting enzyme may be recovered from the medium by conventional procedures including separating the cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, followed by purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, gel filtration

chromatography, affinity chromatography, or the like.

Preferably, the isolated protein is about 90% pure as determined by SDS-PAGE, purity being most important in food, juice or detergent applications.

In a particularly preferred embodiment, the expression of laccase is achieved in a fungal host cell, such as

Aspergillus . As described in detail in the following examples, the laccase gene is ligated into a plasmid containing the Aspergillus oryzae TAKA α-amylase promoter, and the Aspergillus nidulans amdS selectable marker.

Alternatively, the amdS may be on a separate plasmid and used in co-transformation. The plasmid (or plasmids) is used to transform an Aspergillus species host cell, such as A. oryzae or A., niger in accordance with methods described in Yelton et al. (PNAS USA 81: 1470-1474,1984).

It is of particular note that the yields of Polyporus laccase in the present invention, using Aspergillus as host cell are unexpectedly and considerably higher than has previously been reported for expression of other laccases in other host cells. It is expected that the use of

Aspergillus as a host cell in production of laccases from other basidiomycetes, such as Coriolus or Trametes, will also produce larger quantities of the enzyme than have been previously obtainable. The present invention therefore also encompasses the production of such Polyporus-like laccases in Aspergillus recombinant host cells.

Those skilled in the art will recognize that the invention is not limited to use of the nucleic acid

fragments specifically disclosed herein, for example, in Figures 1-5. It will also be apparent that the invention encompasses those nucleotide sequences that encode the same amino acid sequences as depicted in Figure 1-5, but which differ from the specifically depicted nucleotide sequences by virtue of the degeneracy of the genetic code. Also, reference to Figures 1-5 in the specification and the claims will be understood to encompass both the genomic sequence depicted therein as well as the corresponding cDNA and RNA sequences, and the phrases "DNA construct" and "nucleic acid sequences" as used herein will be understood to encompass all such variations. "DNA construct" shall generally be understood to mean a DNA molecule, either single- or double- stranded, which may be isolated in partial form from a naturally occurring gene or which has been modified to contain segments of DNA which are combined and juxtaposed in a manner which would not otherwise exist in nature. In addition, the invention also encompasses other

Polyporus laccases, including alternate forms of laccase which may be found in Polyporus pinsi tus and as well as laccases which may be found in other fungi falling within the definition of Polyporus as defined by Fries, or synonyms thereof as stated in Long et al., 1994, ATCC Names of

Industrial Fungi, ATCC, Rockville, Maryland. Identification and isolation of laccase genes from sources other than those specifically exemplified herein can be achieved by

utilization of the methodology described in the present examples, with publicly available Polyporus strains.

Alternately, the sequence disclosed herein can be used to design primers and/or probes useful in isolating laccase genes by standard PCR or southern hybridization techniques. Other named Polyporus species include, but are not limited to, P. zonatus, P. alveolaris, P. arcularius, P.

australiensis, P. badius, P. biformis, P. brumalis, P.

ciliatus, P. colensoi, P. eucalyptoxrum, P. meridionalis, P. varius, P. palustris, P. rhizophilus, P. rugulosus, P.

squamosus, P. tuberaster, and P. tumulosus . Also

encompassed are laccases which are synonyms, e.g., anamorphs or perfect states of species or strains of the genus

Polyporus . Strains of Polyporus are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), e.g., ATCC 26721, 9385, 11088, 22084, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM),e.g., DSM 1021, 1023, and 1182; and Centraalbureau Voor Schimmelcultures (CBS), e.g., CBS 678.70, 166.29, 101.15, 276.31, 307.39, 334.49, and 332.49. The invention also encompasses any variant nucleotide sequence, and the protein encoded thereby, which protein retains at least about an 80% homology, preferably at least about 85%, and most preferably at least about 90-95%

homology with any one of the amino acid sequences depicted in Figures 2-5, and which qualitatively retains the laccase activity of the sequence described herein. Useful variants within the categories defined above include, for example, ones in which conservative amino acid substitutions have been made, which substitutions do not significantly affect the activity of the protein. By conservative substitution is meant that amino acids of the same class may be substituted by any other of that class. For example, the nonpolar aliphatic residues Ala, Val, Leu, and lie may be

interchanged, as may be the basic residues Lys and Arg, or the acidic residues Asp and Glu. Similarly, Ser and Thr are conservative substitutions for each other, as are Asn and Gin. It will be apparent to the skilled artisan that such substitutions can be made outside the regions critical to the function of the molecule and still result in an active enzyme. Retention of the desired activity can readily be determined by conducting a standard ABTS oxidation method, such as is described in the present examples.

The protein can be used in number of different

industrial processes. These processes include polymerizatio of lignin, both Kraft and lignosulfates, in solution, in order to produce a lignin with a higher molecular weight. Such methods are described in, for example, Jin et al., Holzforschung 45(6): 467-468, 1991; US Patent No. 4,432,921; EP 0 275 544; PCT/DK93/00217, 1992.

The laccase of the present invention can also be used for in-situ depolymerization of lignin in Kraft pulp, thereby producing a pulp with lower lignin content. This use of laccase is an improvement over the current use of chlorine for depolymerization of lignin, which leads to the production of chlorinated aromatic compounds, which are an environmentally undesirable by-product of paper mills. Suc uses are described in, for example, Current opinion in Biotechnology 3: 261-266, 1992; J. Biotechnol. 25: 333-339, 1992; Hiroi et al., Svensk papperstidning .5: 162-166, 1976.

Oxidation of dyes or dye precursors and other

chromophoric compounds leads to decolorization of the compounds. Laccase can be used for this purpose, which can be particularly advantageous in a situation in which a dye transfer between fabrics is undesirable, e.g., in the textile industry and in the detergent industry. Methods for dye transfer inhibition and dye oxidation can be found in WO 92/01406, WO 92/18683, EP 0495836 and Calvo, Mededelingen van de Faculteit Landbouw-wetenschappen/Rijiksuniversitet Gent.56: 1565-1567, 1991; Tsujino et al., J. Soc. Chem.42: 273-282, 1991.

The laccase is particularly well-suited for use in hair dyeing. In such an application, the laccase is contacted with a dye precursor, preferably on the hair, whereby a controlled oxidation of the dye precursor is achieved to convert the precursor to a dye, or pigment producing compound, such as a quinoid compound. The dye precursor is preferably an aromatic compound belonging to one of three major chemical families: the diamines, aminophenols (or aminonaphthols) and the phenols. The dye precursors can be used alone or in combination. At least one of the

intermediates in the copolymerization must be an ortho- or para-diamine or aminophenol (primary intermediate). Examples of such are found in Section V, below, and are also

described in US Patent No. 3,251,742, the contents of which are incorporated herein by reference. In one embodiment, the starting materials include not only the enzyme and a primary intermediate, but also a modifier (coupler) (or combination of modifiers), which modifier is typically a meta-diamine, meta-aminophenol, or a polyphenol. The modifier then reacts with the primary intermediate in the presence of the laccase, converting it to a colored compound. In another embodiment, the laccase can be used with the primary intermediate directly, to oxidize it into a colored compound. In all cases, the dyeing process can be conducted with one or more primary intermediates, either alone or in combination with one or more modifiers. Amounts of components are in accordance with usual commericial amounts for similar components, and proportions of

components may be varied accordingly.

The use of this laccase is an improvement over the more traditional use of H₂O₂, in that the latter can damage the hair, and its use usually requires a high pH, which is also damaging to the hair. In contrast, the reaction with laccase can be conducted at alkaline, neutral or even acidic pH, and the oxygen needed for oxidation comes from the air, rather than via harsh chemical oxidation. The result provided by the use of the Polyporus laccase is comparable to that achieved with use of H₂O₂, not only in color

development, but also in wash stability and light fastness.

An additional commercial advantage is that a single

container package can be made containing both the laccase and the precursor, in an oxygen free atmosphere, which arrangement is not possible with the use of H₂O₂.

The present laccase can also be used for the

polymerization of phenolic or aniline compounds present in liquids. An example of such utility is the treatment of juices, such as apple juice, so that the laccase will accelerate a precipitation of the phenolic compounds present in the juice, thereby producing a more stable juice. Such applications have been described in Stutz, Fruit processing 7/93, 248-252, 1993; Maier et al., Dt. Lebensmittel- rindschau 86(5) : 137-142, 1990; Dietrich et al., Fluss. Obst 57(2): 67-73, 1990.

Laccases such as the Polyporus laccase are also useful in soil detoxification (Nannipieri et al., J. Environ. Qual. 20: 510-517,1991; Dec and Bollag, Arch. Environ. Contam. Toxicol. 19: 543-550, 1990).

The invention is further illustrated by the following non-limiting examples.

EXAMPLES

I. ISOLATION OF A POLYPORUS PINISITUS LACCASE ENZYME

MATERIALS AND METHODS

1. Enzymatic assays

Unless otherwise stated, throughout the examples, laccase activity is determined by syringaldazine and 2,2'- bisazino (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), as follows. The oxidation of syringaldazine is monitored at 530 nm with 19 μM substrate. In 25 mM sodium acetate, 40 μM cupric sulfate, pH 5.5, at 30°C, the activity is expressed as LACU(μmole/min). For pH profile studies, Britton &

Robinson (B&R) buffers are used, and are prepared according to the protocol described in Quelle, Biochemisches

Taschenbuch, H.M. Raven, II. Teil, S.93 u. 102, 1964. ABTS oxidation is carried out with ImM ABTS in 0.1 M NaAc, pH 5.0 at room temperature by monitoring either ΔAbs₄₀₅ in a 96-well plate (Costar) or ΔAbs₄₁₈ in a quartz cuvette. The overlay

ABTS oxidase activity assay is carried out by pouring cooled ABTS-agarose(0.03-0.1 g ABTS, 1 g agarose, 50 ml H₂O, heated to dissolve agarose) over a native IEF gel or PAGE and incubating at room temperature.

2. Initial isolation of laccase

In order to isolate the laccase, 800 ml of culture fluid is filtered by HFSC on a Supra filter(slow filtering). The clear filtrate is then concentrated and washed on an Amicon cell with a GR81 PP membrane to a volume of 72 ml.

One ml aliquots of laccase are bound to a Q-sepharose HP (Pharmacia, Sweden) column, equilibrated with 0.1 M phosphate, pH7 and the laccase is eluted with a NaCl gradient. In all, 10 x 1 ml samples are purified, pooled, concentrated and washed by ultrafiltration using a membrane with a molecular weight cut-off of 6kD.

3. Secondary purification

In a second purification, a fermentation broth is filtered and concentrated by ultrafiltration. The starting material contains 187 LACU/ml. The concentrate is quick- filtered on a Propex 23 filter (P & S Filtration), with 3% Hyflo Cuper-Cel (HSC; Celite Corporation), followed by two ultrafiltration on a Filtron filter with two membranes, each with a molecular weight cutof of 3 kD. The resulting sample (2.5 mS/cm, pH 7.0, at 4°C) is applied to a 130 ml Q- Sepharose column, equilibrated with sodium phosphate, 1.1 mS/cm, pH 7.0. Under these conditions the laccase does not bind to the column, but elutes slowly from the column during the application and wash with the equilibration buffer, resulting in a partial separation from other brownish material.

This partially purified preparation of 1.0mS, pH 7.0 at 20°C is applied to a Q-sepharose column. The column is equilibrated with 20mM sodium phosphate, 2.2 mS, pH 7.0.

Under these conditions, the laccase binds to the column and is eluted by a gradient of 0-1 M NaCl over 20 column

volumes.

3. Sequencing

For internal peptide sequencing, the purified protein is digested with trypsin, followed by peptide purification with HPLC. Purified peptides are sequenced in an Applied Biosystems 473A sequencer.

B. RESULTS AND DISCUSSION

1. Initial characterization

Total yield of the initial purification is about 50 mg (estimated at A280nm). The purified enzyme has a rich blue color, and appears as only two very close bands on SDS- PAGE at about 65 kd. A native PAGE overlaid with substrate shows that both bands have laccase activity with ABTS. The absorption spectrum shows that besides an absorption at A280nm, the purified laccase also shows absorption at about 600nm.

2. Sequencing

A N-terminal determination of the protein initially purified shows a single sequence:

Gly-Ile-Gly-Pro-Val-Ala-Asp-Leu-Thr-Ile-Thr-Asn- Ala-Ala-Ala-Val-Ser-Pro-Asp-Gly-Phe-Pro...

Since the N-terminal sequence is not the ideal sequence for constructing a probe, additional experiments with a trypsin digest are conducted, followed by further

purification (described above) and sequencing of fragments

2. Secondary purification and characterization

In the second purification, the second Q-Sepharose chromatographic step yields the following pools:

Q-Sepharose-2-pool-1 40 ml 112 LACU 47 LACU/A₂₈₀

Q-Sepharose-2-pool-3 80 ml 385 LACU 65 LACU/A₂₈₀

The elution yields >80% of the applied amount. The highly purified preparation Q-Sepharose-2-pool-3 has an A₂₈₀ = 5.9, and A₂₈₀/A₂₆₀ = 1-4. The purity of the laccase in the

starting material is extremely high on a protein basis but the starting material is a very dark brown color. In SDS- PAGE, a double band is seen, with a dominating 65 kD band and a smaller 62 kD band. By anionic chromatography, only the dominating band is seen in the first peak (Q-Sepharose-2- pool-1), whereas both bands are seen in the second peak(Q- Sepharose-2-pool-3).

3. Sequence

A number of internal peptide sequences are determined, and compared with the Coriolus hirsutus (Ch) laccase

sequence. The identified fragments are as follows:

Tryp 13:

Ser-Pro-Ser-Thr-Thr-Thr-Ala-Ala-Asp-Leu Tryp 14:

Ser-Ala-Gly-Ser-Thr-Val-Tyr-Asn-Tyr-Asp-Asn-Pro-Ile-Phe Arg

Tryp 16:

Sequence 1:

Ser-Thr-Ser-Ile-His-Trp-His-Gly-Phe-Phe-Gln-Lys

Sequence 2:

Gly-Ile-Gly-Pro-Val-Ala-Asp-Leu-Thr-lle-Thr-Asn-Ala-Ala-Val

Tryp 18:

Gly-Ile-Gly-Pro-Val-Ala-Asp-Leu-Thr-lle-Thr-Asn

Tryp 19:

Sequence 1:

Leu-Gly-Pro-Ala-Phe-Pro-Leu-Gly-Ala-Asp-Ala-Thr-Leu-Ile-

Sequence 2:

Phe-Gln-Leu-Asn-Val-Ile-Asp-Asn-Asn-Thr-Thr-His-Thr-Met Tryp 25:

Tyr-Ser-Phe-Val-Leu-Glu-Ala-Asn-Gln-Ala-Val-Asp-Asn-Tyr-Trp-

Ile-Arg

Tryp 27

Gly-Thr-Asn-Trp-Ala-Asp-Gly-Pro-Ala-Phe

II. ISOLATION OF A POLYPORUS PINISITUS LACCASE CDNA CLONE A. MATERIALS AND METHODS

1. RNA preparation

RNA is isolated from 10 grams of P. pinsitus mycelium grown under xylidine induction for 6.5 hours, using the guanidium/CsCl cushion method. The RNA is poly-A selected on an oligo-dT column, using standard conditions. 120μg mRNA is obtained and stored as lyophilized pellet in 5μg aliquots at -80°C.

2. Single stranded cDNA

Single stranded cDNA is synthesized using the reverse transcriptase "Super Script" (BRL) according to

manufacturer's directions.

3. Construction of cDNA library A cDNA library is constructed using the librarian IV cDNA kit (Invitrogen). Fifty cDNA pools, each containing approximately 5000 individual transformants, are obtained.

4. PCR

PCR is conducted under the following standard

conditions: 100pmol of each primer, 10μl 10X PCR

buffer (Perkin-Elmer), 40μl dNTP 0.5 mM, 2μl single stranded cDNA(or approximately 100 ng chromosomal DNA or 100 ng PCR fragment), H₂O to 100 μl, 2.5U Taq polymerase. The cycles are 3x(40°C/two minutes, 72°C/two minutes, 94°C/one minute) followed by 30x(60°C/two minutes, 72°C/two minutes, 94°C/1 minute).

B. RESULTS AND DISCUSSION

1. Cloning of Polyporus vinsi tus laccase

PCR is carried out with the primer #3331:

ACCAGNCTAGACACGGGNTC/AGATACTG/ACGNGAGAGCGGAC/TTGCTGGTC

ACTATCTTCGAAGATCTCG

and primer #3332:

CGCGGCCGCTAGGATCCTCACAATGGCCAA/CTCTCTG/CCTCG/ACCTTC .

A clear band of about 1500bp is obtained. The DNA is digested with Notl/Hindlll, and fractionated on an agarose gel. The upper band (fragment #42) is purified and cloned into the Aspergillus vector pHD423. No transformants are obtained. Several attempts are carried out in order to clone the fragment, including redigestion with the

restriction enzymes, phosphorylation of the ends, filling in with klenow and blunt-end cloning in Smal cut puC18, without success. Hybridization with a laccase probe based on the laccase described in Coll et al., supra, indicates that the PCR product could be the P. pinsitus laccase. In a new attempt to clone the PCR fragment, a new PCR reaction is carried out, using the same conditions as for fragment #42. Again the result is a fragment of about 1500 bp(fragment #43). This time the fragment is cut with HindiII/BamHI, and ligated to HindiII/BamHI-cut pUClδ. Three clones, #43-/A,- B,-G are found to contain a fragment of 1500 bp. Partial sequencing reveals that these fragments are laccase related.

2.Expression of Polyporus pinsitus laccase

To express the laccase, the fragment #43 is joined to a signal sequence from a 43kD cellulase. The primer pHD433 (TAGCGGATCCCACAATGCGTTCCTCCCCCCTCCTCCCGTCCGCCGTTGTGGCCGCCCTG CCGGTGTTGGCCCTTGCCGGCATTGGGCCCGTCGCGGACC) is used in a standard PCR reaction with a pUC forward primer (New England Biolabs). All three clones are used as templates in order to minimize the risk of working with DNA containing errors.

The PCR generated DNA from the reaction with a primer PHD433 and template 43-A and 43-G is cut with Hindlll/BamHI and cloned into the Aspergillus expression vector

pHD414 (described in detail below). Several transformants are obtained.

Clones pHD433/43A-1,2, pHD433/43G-2, -3 are transformed into A. oryzae. The transformants from each transformation (between 3-10) are analyzed for laccase production.

Activity is only obtained with pHD433/43G-3. The positive transformants (numbers 1, 4, 6) are reisolated on amdS plates, and retested. In an additional transformation round a further ten transformants are obtained with pHD433/43G-3. The clones #20, 23, 26, 28, and 29 are positive. The clones are reisolated and two single isolates are analyzed for laccase expression semiquantitatively by color development in an ABTS assay at pH 4.5. On a scale of +-+++, several clones show moderate to strong expression of laccase.

Further cloning is conducted to identify a full length clone. A xylidine-induced cDNA library consisting of approximately 350,000 transformants is screened using fragment #42-4 as a probe. More than 100 positive clones are detected. The clones are purified, rescreened, and analyzed on Southern blots. Two of the longest clones are further characterized by DNA sequence determination. The longest clones are found to be identical and found to contain a poly-A stretch in the 3'end and to start at the amino acid number 4 in the amino terminus. A partial DNA sequence is determined from different clones.

pHD433/43G-3 is then used in further cloning studies as described in the following Section IV.

III. PURIFICATION AND CHARACTERIZATION OF ADDITIONAL

POLYPORUS PINSITUS LACCASE WILD-TYPE ENZYMES

A. MATERIALS AND METHODS

1. Culture conditions

Shake flasks (250 ml medium/2.8 1 baffled flask) are inoculated wtih several agar plugs taken from a week-old PDA plate of P. pinsitus. The medium contains, per liter, 10 g glucose, 2.5 g L-asparagine, 0.2 g L-phenylalanine, 2.0 g yeast extract, 2.0 g KH₂PO₄, 0.5 g MgSO₄·7H₂O, 2.0 mlAMG trace metals, 0.002 g CuSO₄-7H₂O, 1.0 g citric acid, made with tape water, pH 5.0 before autoclaving. The cultures are grown at 18-22°C on a rotary shaker with low agitiation (-100 rpm). After 7 days, the pH of each shake flask is adjusted to -6.0 by the addition of 0.25 ml 5 N NaOH and the cultures are induced by adding 0.5 ml of a 2,5-xylidine stock

solution (xylidine diluted 1:10 into ethanol) to each flask. Flasks are incubated for an additional 24 hours, at which time the culture supernatant from each flask is recovered.

2. Materials.

Chemicals used as buffers are commercial products of at least reagent grade. Endo/N-glucosidase F is from

Boehringer-Mannheim. Chromatography is performed on

Pharmacia FPLC. Spectroscopic assays are conducted on either a spectrophotometer (Shimadzu PC160) or a microplate reader (Molecular Devices).

3. Purification Culture broth is filtered first on cheesecloth and centrifuged at 1000 x g to remove gelatinous pinkish

xylidine polymer. The supernatant is then filtered on

Whatman #2 paper and concentrated from 1500 to 250 ml on SlYlOO (Amicon, Spiral concentrator) at 4°C. The

concentrated broth is diluted with water until it reaches 0.8 mS(from 2.5 mS) and then concentrated on SlY100 to 250 ml. The washed broth, thawed from -20 °C freezing overnight, is subjected to Whatman #2 paper filtration to remove residual pinkish material, and then pH adjusted by NaOH from pH 6.1 to pH 7.7. This yellowish broth, 275 ml with 0.8 mS, is applied on a Q-Sepharose XK-26 column (-64 ml gel) equilibrated with 10 mM Tris-HCl, pH 7,7, 0.7 mS. The first active laccase fraction runs through during loading and washing by the equilibrating buffer. The elution is carried out by a linear gradient of 0-0.5 M NaCl in the

equilibrating buffer over 8.8 bed-volume. The second and third active fractions are eluted around 0.15 and 0.35M NaCl, respectively. No more active fractions are detected when the column is washed sequentially with 2 M NaCl and with 1 mM NaOH. The active fractions are pooled, adjusted to ~10mS, concentrated on Centricon-10 (Amicon), and then applied onto Superdex 75(HR10/30, 24 ml, Pharmacia)

equilibrated with 10mM Tris-HCl, 0.15 M NaCl, pH 8, 14 mS. During elution with the application buffer, laccase

fractions are eluted off using the same elution volume for all three Q-Sepharose fractions, indicating very similar native molecular weight. The purity of the laccase is tested on SDS-PAGE.

4. Protein analysis

PAGE and native IEF are carried out on a Mini Protean II and a Model 111 Mini IEF cells (Bio-Rad). Western blots are carried out on a Mini trans-blot cell (Bio-Rad) with an alkaline phosphatase assay kit (Bio-Rad). The primary antibodies are diluted 1000-fold during blotting. N- terminus sequencing is performed on an Applied Biosystems (ABI) 476A protein sequencer using liquid phase TFA delivery for cleavage and on-line HPLC for identification of PTH- amino acids. Standard Fast Cycles and Pre-Mix Buffer System is used according to manufacturer's instructions.

Deglycosylation with glycosidase is done as follows: 3μg of protein and 3.6 units of glycosidase in 0.25M NaAc, pH 5, 20 mM EDTA, 0.05% 2-mercaptoethanol is incubated at 37°C for 18 hours with ovalbumin and bovine serum albumin serving as positive and negative control, respectively, and the

mobility is detected by SDS-PAGE.

Amino acid analysis for determining extinction

coefficients is done using Amino Quant 1090 HPLC system from Hewlett Packard. Microwave facilitated vapor phase

hydrolysis of lyophilized samples is done using the MDS-2000 hydrolysis-station (CEM, Matthews, NC). 6N HCl containing 1% phenol as a scavenger is used to create the acid vapors. Hydrolysis time is 20 minutes at 70 psi (-148°C).

Hydrolyzed samples are lyophilized and redissolved in 20 μl of 500pmol/μl sarcosine and norvaline as internal standards. lμl is injected and analyzed according to manufacturer's instructions.

B. RESULTS AND DISCUSSION

1. Purification

The previously characterized P. pinsitus laccase has a pi of -3.5. However, considerable laccase activity is detected in the run-through fraction of Q-Sepharose pre- equilibrated at pH 7.7. Upon a gradient elution, one more active fraction comes off the column before the active fraction initially anticipated. UV-visible spectra and SDS- PAGE show that all three fractions contain mainly laccase. After further purification by gel filtration, different pl's under native non-denaturing conditions are detected for the two new fractions and shown to be consistent with the elution order.

2. Characterization

The pure laccase preparations derived from Q-Sepharose eluates behave as a rather well-defined band on SDS-PAGE at -63 kDa. Deglycosylation detects -14% w/w carbohydrates based on mobility change on SDS-PAGE. On native-IEF, the laccase preparations have bands of pi 6-6.5, 5-6.5, and 3.5. ABTS-agarose overlay show that all bands are active. Each form in turn shows multiple isoforms under the IEF

conditions.

The neutral and acidic forms have a typical UV-visible spectrum with maxima at 605 and 275 nm. The ratio of

A₂₇₅/A₆₀₅ is 30-40. The spectrum for the acidic-neutral form has a peak at 276 nm and a shoulder around 600 nm.

The N-terminal sequencing shows that the neutral and neutral-acidic forms have the same first 29 residues (Table 1). The N-terminus of the acidic form matches 100% to that of the previously characterized form. All three forms exhibit comparable cross-reactivity toward antibodies raised against previously characterized form.

3. Laccase Activity

The specific activities (per A₂₇₅) of the three forms are tested by both ABTS and syringaldazine oxidations. The shapes and optima of the pH activity profiles for the three forms are very close: all have optima at ≤pH4 and pH 5-5.5 for ABTS and syringaldazine oxidations, respectively.

IV. ISOLATION OF MULTIPLE COPIES OF POLYPORUS PINSITUS LACCASE ENZYMES AND GENES

A. MATERIALS AND METHODS

1. Strains

The following strains are employed in the methods described below: E. coli K802 (el4-(mrca), mcrB, hsdR2, galK2, galT22, supE44, metB1; Clonetech); E. coli XL-1

Blue (recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1, lac[F'proAB, lacIqZDM15, Tn10 (tetr)];Stratagene) and

Polyporus pinsitus CBS 678.70.

2. Genomic DNA isolation

Cultures of P.pinsitus are grown in 500 ml YG (0.5% yeast extract, 2% dextrose) at room temperature for 3 to 4 days. Mycelia are harvested through miracloth, washed twice with TE and frozen quickly in liquid nitrogen. The frozen mycelia are stored at -80°C. To isolate DNA, the mycelia are ground to a fine powder in an electric coffee grinder. The powdered mycelia are resuspended in TE to a final volume of 22 ml. Four ml 20% SDS is added with mixing by inversion followed by incubation at room temperature for 10 minutes. The sample is gently extracted with phenol: chloroform and centrifuged to separate the phases. The aqueous phase is collected and 400μl proteinase A(10 mg/ml stock) is added. The sample is incubated at 37°C for 30 minutes followed by a phenol: chloroform extraction. The aqueous phase is

precipitated by the addition of 0.1 volumes of 3 M Na acetate, pH 5.2 and 2.5 volumes 95% ethanol and freezing at 20°C for one hour. After centrifugation to precipitate the DNA, the pellet is resuspended in 6 ml TE, and 200 μl boiled RNase A(10 mg.ml stock) is added. After incubation at 37°C, 100 μl proteinase A (10 mg/ml stock) is added followed by incubation at 37°C for 30 minutes. The sample is

phenol : chloroform extracted twice. To the aqueous phase, 0.1 volumes 3 M Na acetate and 2.5 volumes are added, and teh sample is frozen at -20°C for 1 hour. Following

centrifugation, the pellet is gently resuspended in 400 μl TE, and 40 μl Na acetate and 1 ml 95% ethanol are added. The DNA is pelleted by centrifugation, and the pellet is washed in 70% ethanol. The final pellet is resuspended in 250 μl TE.

3. RNA preparation

RNA is isolated from mycelia which are harvested from P. pinisitus cultures which are either induced for laccase expression by the addition of 2,5-xylidine or are uninduced. The mycelia are washed and frozen quickly in liquid N₂.

Frozen mycelia are ground to a fine powder in an electric coffee grinder. The powder is immediately suspended in 20 ml extraction buffer (0.2 M Tris-HCl, 0.25 M NaCl, 50 mM EGTA, 0.8% tri-isopropyl naphthalene sulfonic acids, 4.8% p- aminosalicylic acid, pH 8.5). All solutions for RNA extraction are made with diethylpyrocarbonate (DEP) -treated water. The sample is kept on ice and 0.5 volumes TE- saturated phenol: chloroform is added. The sample is mixed well by inversion for 2 minutes, and the phases are

separated by centrifugation. The aqueous phase is saved, and the organic phase is extracted with 2 ml extraction buffer and incubated at 68°C for 5 minutes. After

centrifugation to separate the phases, the aqueous phases are pooled and extracted several time with phenol: chloroform until there is no longer any protein at the interface. To the aqueous phase 0.1 volume 3 M Na-acetate, pH 5.2 and 2.5 volumes 95% ethanol are added to precipitate the RNA, and the sample is frozen at -20°C for 2 hours. The RNA is pelleted and resuspended in DEP water with RNase inhibitor.

4. DNA sequencing

Nucleotide sequences are determined using TAQ

polymerase cycle sequencing with fluorescent-labeled nucleotides, and reactions are electrophoresed on an Applied Biosystems automatic DNA sequencer (Model 363A, version 1.2.0).

5. Preparation of genomic libraries

Two size-selected genomic libraries of P. pinsitus are constructed. A library of 5 to 6 kb BamHI fragments are constructed in pBluescript+. Genomic DNA is digested with BamHI, and the digest is electrophoresed on a preparative agarose (IBI) gel. The region containing the 5 to 6 BamHI fragments is sliced from the gel. The DNA is isolated from teh gel using a Geneclean kit (BIO 101). The DNA is ligated into pBluescript plasmid previously digested with BamHI and dephosphorylated with BAP(GIBCO BRL), E. coli XL-1 Blue competent cells (Stratagene) are transformed with the ligation, and 12,000 white colonies are obtained.

A library of 7 to 8 kb BamHI/EcoRI fragments is constructed in pUC118. Ten μg genomic DNA is digested with BamHI and EcoRl and treated with BAP(GIBCO BRL). Competent E. coli XL-1 Blue cells are transformed with the ligation, and the library contains -8000 recombinants.

For the preparation of a total genomic library in lambda EMBL4, 25 μg of P. pinsitus genomic DNA is partially digested with Sau3A. After digestion, the DNA is

electrophoresed on a preparative low-melt agarose gel, and a band containing the 9 to 23 kb sized DNA is sliced from the gel. The DNA is extracted from the gel using β-agarose (New England Biolabs). The isolated EMBL4 arms (Clonetech) according to the supplier's directions. The ligation. is packaged in vitro using a Gigapack II kit (Stratagene). The library is titered using E. coli K802 cells. The

unamplified library is estimated to contain 35,000

independent recombinants. The library is amplified using E. coli K802 cells.

6. Southern and Northern Blots

DNA samples are electrophoresed on agarose gels in TAE buffer using standard protocols. RNA samples are electrophoresed on agarose gels containing formaldehyde. Both DNA and RNA gels are transferred to Zeta-Probe

membrane (BIO-RAD) using either capillary action under alkaline conditions or a vacuum blotter. After transfer, the DNA gels are UV crosslinked. Blots are prehybridized at 65°C in 1.5X SSPE, 1% SDS, 0.5% non-fat dried milk and 200 μg/ml salmon sperm DNA for 1 hour. Radioactive probes are added directly to the prehybridization solutions, and hybridizations are continued overnight at 65°C. Blots are washed with 2XSSC for 5 minutes at 65°C and with 0.2XSSC, 1%SDS,0.1% Na-pyrophosphate at 65°C for 30 minutes twice.

Radioactive labeled probes are prepared using a α-³²P- dCTP and a nick translation kit (GIBCO-BRL).

7. Library screening For screening of the size-selected 5-6 kb BamHI and 7-8 kb BamHI/EcoRl libraries -500 colonies on LB carb plates and lifted the colonies to Hybond N⁺ filters (Amersham) using standard procedures. The filters are UV crosslinked

following neutralization. The filters are prehybridized at 65°C in 1,5X SSPE, 1% SDS, 0.5% non-fat dried milk, 200 μg/ml salmon sperm DNA for 1 hour. Nick-translated probes are added directly to the prehybridization solution, and hybridizations are done overnight at 65°C.

For screening of the genomic bank in EMBL, appropriate dilutions of the amplified library are plated with E. coli K802 cells on 100mM NZY top agarose. The plaques are lifted to Hybond N⁺ membranes (Amersham) using standard procedures. The DNA is crosslinked to the membranes using UV

crosslinking. The filters are prehybridized and hybridized using the same conditions as those mentioned above.

RESULTS AND DISCUSSION

1. Isolation of multiple copies of laccase gene

P. pinsitus genomic DNA is digested with several different restriction enzymes for southern analysis. The blot is probed with the cDNA insert (isolated as a BamHi/Sphi fragment from the pYES vector) which is labeled with α-P³²- dCTP. The blot is hybridized and washed as described above. The cDNA hybridizes to several restriction fragments for most of the enzymes suggesting that there are multiple laccase genes in the genome. Because the cDNA hybridizes to a BamHI fragment of -5.5 kb, a library of 5-6 kb BamHI fragments from P. pinisitus is constructed.

2. Screening of Genomic Libraries

The results from screening of the libraries are summarized in Table 2. The 5-6 kb BamHI size-selected library is screened with the original cDNA clone labeled with ³²P. Approximately 30,000 colonies are screened with hybridizations done at 65°C. Plasmid DNA is isolated from two positive colonies and digested with BamHI to check for insert size. Both clones contain an -5.5 kb BamHI insert. The cloned insert (LCC3) is sequenced from either end; the sequence has homology to the cDNA, but is clearly not the cDNA encoded laccase. The partial DNA sequence of LCC3 also indicates that the LCC3 pUC118 clone does not contain the full gene.

From a southern blot of BamHl/EcoRl double digested DNA it is demonstrated that the cDNA hybridizes to an -7.7 kb fragment. A size-selected library in pUC118 is constructed containing 7-8 BamHI/EcoRI fragments. A total of -8000 independent colonies are obtained and screened by

hybridization with a ³²P labeled insert. Plasmid DNA is isolated from the positive colonies and digested with BamHI and EcoRl. Restriction analysis of the plasmids demonstrate that they fall into two classes. One class (LCC4) contains four clones which are all identical and have an -7.7 kb BamHI/EcoRI insert which hybridizes to the cDNA. A second class (LCC1) contains two clones which are identical and have inserts of -7.2 kb which hybridize to the cDNA. Partial DNA sequencing of clones LCC1 and LCC4 demonstrate that clone 21 is the genomic clone of the original cDNA, while LCC4 codes for another laccase. The partial DNA sequence of LCC1 shows that the pUCllδ clone does not contain the full gene and that a fragment upstream of the EcoRl site is needed.

At the same time the size selected 7-8 BamHI/EcoRI library is being constructed, a P. pinisitus genomic bank in EMBL4 is constructed containing -35,000 independent

recombinant phage. Ten positive plaques are picked and purified. DNA is isolated from the purified phage lysates. Restriction digests of EMBL DNAs demonstrates that there are three classes of clones. The first class (11GEN) is defined by two sibs whose inserts contain a BamHI/EcoRI fragment of the same size as LCC1 which hybridizes to the LCC1 insert. The second class (12GEN) contains one clone which has a different restriction pattern than the 11GEN class and whose insert contains a different restriction pattern than the 11GEN class and whose insert contains an -5.7 kb BamHI/EcoRI fragment. The third class is defined by a single clone whose insert contains an -3.2 kb BamHI/EcoRI fragment which hybridizes to the LCC1 insert. DNA sequence analysis demonstrates that clone 11GEN contains the LCC1 BamHI/EcoRI fragment and both 5' and 3" flanking regions. It is also demonstrated that clone 12GEN contains a portion of the LCC1 insert.

The P. pinisi tus EMBL genomic bank is also screened with the LCC3 BamHI insert in order to clone the full gene. Approximately 30,000 plaques are plated and lifted from hybridization. Five plaques which hybridize to the

LCC3 (BamHI/EcoRI) insert are identified and purified. DNA is isolated from the purified phage stocks. Southern analysis of P. pinisitus genomic DNA demonstrates that the LCC3 BAmHI insert hybridizes to an ~7kb EcoRl fragment.

Restriction digests and southerns demonstrate that 4 of the clones contain restriction fragments which hybridize to the EcoRI/BamHI (1.6 kb) fragment and that the clones fall into three classes. Class one is defined by a single clone (LCC5) whose insert contains a 3kb EcoRl fragment which hybridizes to the LCC3 BamHI/EcoRI fragment. Another class is defined by clone (LCC2) whose insert contains an -11 kb EcoRl fragment which hybridizes to the LCC3 BamHI/EcoRI insert. The third class is defined by two clones which are not identical but contain many of the same restriction

fragments; these clones both contain an -7.5 kb EcoRl fragment which hybridizes to the LCC3 insert. Further analysis of this third class indicates that they are identical to clone LCC4. Partial DNA sequencing of LCC5 and LCC2 indicates that both of these clones code for laccases; however, neither is identical to any of the above mentioned laccase genes (LCC1, LCC3, or LCC4). At this point, five unique laccase genes are cloned; however, the fragments subcloned from LCC5 and LCC2 do not contain the full genes.

From the DNA sequencing of the 3 kb EcoRl fragment from clone LCC5 it is determined that -200 base pairs of the N- terminus are upstream of the EcoRl site. A 380 bp

EcoRI/MluI fragment from LCC5 is used to identify for subcloning a Mlul fragment from the LCC5 EMBL clone. An -4.5 Mlul fragment from the LCC5 EMBL clone is subcloned for sequencing and shown to contain the N-terminal sequence.

To clone the N-terminal half of the LCC3 laccase gene, the P. pinsitus EMBL genomic bank is probed with an -750 bp BamHI/StuI restriction fragment from the LCC3 pUCllδ clone. Approximately 25,000 plaques are screened and five plaques appear to hybridize with the probe. Upon further

purification only three of the clones are still positive. Two of the clones give very strong signals and the

restrictions digests of DNA isolated from these phage demonstrate that both contain an -750 bp BamHI/StuI fragment in their inserts and that the two clones are not identical but overlapped. Based on results of Southern analysis, an -8.5 kb fragment from these clones are subcloned for sequencing. The EcoRl fragment is shown to contain the entire gene.

To clone the N-terminal half of the LCC2 laccase gene, the P. pinsitus genomic bank in EMBL4 is probed with an -680 bp EcoRI/PvuI of the EMBL LCC2 clone. Thirty thousand plaques are screened by hybridization at 65°C, and 15 plaques appear to hybridize with the probe. All fifteen are purified, and DNA is isolated. The clones can be placed in four classes based on restriction patterns, Seven of the clones are all sibs, and are identical to the original EMBL clone of LCC2. The second class is defined by 3 clones which are sibs. An -4 kb Hindlll fragment is subcloned from this class for sequencing and is shown to contain the N- terminal half of LCC2. A third class is defined by a single clone and is not characterized further.

3. DNA sequencing

The complete DNA sequences of the five genomic clones is determined as described in Materials and Methods.

Sequencing of clone LCC2 demonstrate that it probably codes for the second form of laccase (neutral pI) isolated from culture broth from an induced P. pinsitus culture as described above. The N-terminal protein sequence from the neutral pl laccase and the predicted N-terminus for the protein coded for by LCC2 are compared, and show identity. The predicted pi for the protein coded for by clone LCC2 is 5.95, which is in good agreement with the experimental pi determined for the second form of laccase being between 5.0 and 6.5. Figures 1-5 (SEQ ID NOS. 1-5) show the DNA sequences and predicted translation products for the genomic clones. For LCC1, the N-terminus of the mature protein as determined by protein sequencing and predicted by Von Heijne rules is Gly at position 22. The N-terminus is Gly-Ile-Gly- Pro-Val-Ala-. For LCC2 the N-terminal amino acid of the mature protein as determined by protein sequencing is Ala at position 21. The N-terminus is Ala-Ile-Gly-Pro-Val-Ala-. For LCC3 the predicted N-terminal amino acid of the mature protein is Ser at position 22, with the N terminus being Ser-Ile-Gly-Pro-Val-Thr-Glu-Leu-. For LCC4, the predicted N-terminal amino acid is Ala at position 23 with the N- terminuε being Ala-Ile-Gly-Pro-Val-Thr-. For LCC5 the predicted N-terminal amino acid is Ala at position 24 with the N-terminus being Ala-Ile-Gly-Pro-Val-Thr-Asp. A comparison of the structural organization of the genes and the predicted proteins they code for is presented in Table 1. It will be seen that the five genes have different structural organizations and code for proteins of slightly different sizes. Comparisons between the predicted proteins of the genomic clones and other fungal laccase are also done. Table 2 shows a comparison of the predicted laccase to each other and to other fungal laccases. Clone LCC1 (the induced laccase first characterized) has the most

identity (90%) to the Coriolus hirsutus laccase and the PMl basidiomycete laccase (Coll et al., supra) . The other four laccases have between 64 and δ0% identity to the C. hirsutus laccase. The laccase coded for by LCC3 has the least identity to the LCC1 laccase and the other fungal laccases shown in Table 2. LCC2 appears to be the second wild-type laccase isolated as described above; based on the N-terminal sequences of the isolated clones, it also appears that the "neutral" and acidic neutral" wild-type laccases are the same enzyme which is encoded by the LCC2 sequence.

5. Northern blots

RNA is isolated from mycelia from both a xylidine- induced culture and an uninduced culture. RNA is blotted to membrane after electrophoresis, and the blot is probed with the cDNA insert, or a small fragment containing -100 bp of the 23GEN promoter and the first 100 bp of the coding region. A transcript of about 1.8 kb hybridizes to both the induced and uninduced RNA samples; however, transcription of this message is clearly induced by the addition of xylidine to the culture.

III. EXPRESSION OF P. PINSITUS LACCASE IN ASPERGILLUS

MATERIALS AND METHODS

1. Strains

A. oryzae A1560, A. oryzae HowBl04 (fungamyl delete, pyrg) , A . oryzae HowB101pyrg, A. niger Bo-1, A. niger Bo-80, A. niger ATCC1040, A. niger NRRL337, A. niger NRRL326, A. niger NRRL326, A. niger NRRL2295, A . niger ATCC11358, A . niger NRRL322, A. niger AT10864, A. japonicus A1438, A.

phoenicis, A. foetidus N953.

2. Media

For the shake flask cultivation of the A. niger, A. foetidus, and A. phoenicis MY50 (per liter: 50 g

maltodextrin, 2 g MgSO₄.H₂O, 10 gKH₂PO₄, 2 g K₂SO₄, 2 g citric acid, 10 g yeast extract, 0.5 ml trace metals, 2 g urea, pH 6.0) media is used. For the shake flask cultivation of the A. oryzae A1560 and HowBlOl strains MY51(per liter: 30 g maltodextrin, 2 mg MgSO₄, 10 g KH₂PO₄, 2 g K₂SO₄, 2 g citric acid, 10 g yeast extract, 0.5 ml trace metals, 1 g urea, 2 g(NH₄)₂SO₄, pH 6.0) is used. For the shake flask analysis of the A. oryzae HowBl04 strains, MY51 maltose (same as MY51 but with 50g of maltose instead of maltodextrin) media is used. For the shake flask analysis of the A . japonicus strains M400 media (per liter: 50 g maltodextrin, 2 g MgSO₄, 2 g KH₂PO₄, 4 g citric acid, 8 g yeast extract, 0.5 ml trace metals, 2 g urea, pH 6.0.

Cultures grown overnight for protoplast formation and subsequent transformation are grown in YEG(0.5% yeast extract, 2% dextrose). For strains that are pyrg, uridine is supplemented to 10 mM final concentration.

3. Screening for laccase production

Primary transformants are screened first on a minimal medium plates containing 1% glucose as the carbon source and 1mM ABTS to test for production of laccase. Transformants that give green zones on the plates are picked and spore purified before shake flask analysis is done.

Shake flask samples are centrifuged to clear the broth. Dilute or undiluted broth samples are assayed with ABTS

RESULTS AND DISCUSSION

1. Expression in shake flasks

The first expression vector constructed is pDSYl, which contains the TAKA promoter, TAKA signal sequence, P.

pinisitus laccase cDNA beginning at the mature N-terminus and the AMG terminator. The TAKA signal sequence: laccase insert is constructed in 2 steps. First by site directed mutagenesis, an Agel site beginning at bp 107 of the laccase mature coding region is created by a single base change and a Nsil site is created -120 bp downstream of the laccase Stop codon(ACG GGT->ACC GGT and TTC GCT->ATG CAT,

respectively). A small PCR fragment beginning with an Sfil site and ending with the Agel site at 107 bp in laccase is PCR amplified. This fragment contains a piece of the TAKA signal sequence and the first -107 bp of the mature laccase cDNA. Further DNA sequencing of this fragment shows it has a single base change that leads to a substitution of Asn for Thr at position 9 in mature laccase. This substitution creates a potential N-linked glycosylation site. The PCR fragment and Agel/Nsil fragments are cloned into

pMWRl (Figure 6) which has been digested with Sfil/Nsil. The vector pMWRl contains the TAKA promoter, a portion of the TAKA signal sequence which ends with an Sfil site, and the TAKA terminator with a Nsil site inserted directly 5' to the terminator. The resulting expression vector (Figure 7) is used to cotransform several hosts. Methods for co- transformation of Aspergillus strains are as described in Christensen et al., supra .

In the second laccase expression vector, the base change in DSY1 which leads to the substitution of Asn for Thr at amino acid 9 is reverted back to wild type by a PCR reaction. The second expression vector pDSY2 is identical to pDSY1 except for this single base change. Three

different A. oryzae strains and several A. niger strains are cotransformed with pDSY2 and either pTOC90(WO 91/17243) which carries the A . nidulans amdS gene or pS02 which carries the A. oryzae pyrG gene.

Expression of laccase is observed in all hosts tested, with both DSYl and DSY2. Yields range from 0.1-12.0

Δabs/min/ml, with highest yields being observed with A.

niger strains.

A construct pDSY10 is made which contains the TAKA promoter, laccase full-length cDNA including its own signal sequence and the AMG terminator. A 200 bp BamHI/Agel fragment which has a BamHI site immediately 5' to the ATG of the initiation codon and an Agel site at the same position as in pDSY1 is PCR amplified using lacl as template. A Mlul/Hindlll fragment is PCR amplified using pDSY2 as template and begins with the Mlul site present in the cDNA and ends with a Hindu site directly 3' to the stop codon of laccase. The above two fragments and the Agel/Mlul fragment from pDSY2 are ligated into pHD414 to yield pDSY10 (Figure 8).

The vector pHD414 used in expression of laccase is a derivative of the plasmid p775(EP 238 023). In contrast to this plasmid, pHD414 has a string of unique restriction sites between the TAKA promoter and the AMG terminator. The plasmid is constructed by removal of an approximately 200 bp long fragment (containing undesirable RE sites) at the 3' end of the terminator, and subsequent removal of an

approximately 250 bp long fragment at the 5' end of the promoter, also containing undesirable sites. The 200 bp region is removed by cleavage with Narl (positioned in the pUC vector) and Xbal (just 3' to the terminator), subsequent filling in the generated ends with Klenow DNA polymerase + dNTP, purification of the vector fragment on a gel and religation of the vector fragment. This plasmid is called pHD413. pHD413 is cut with StuI (positioned in the 5' end of the promoter) and PvuII (in the pUC vector), fractionated on gel and religated, resulting in pHD414. Cotransformation of A. oryzae HowB104 and A. niger Bo-1 are done using pToC90 for selection. Yields in shake flask are comparable to those seen with pDSY2.

2. Expression in fermentors

A 1 ml aliquot of a spore suspension of Aspergillus niger transformant Bo-1-pDSY10-4 (approximately 10⁹ spores/ml) is added aseptically to a 500 ml shake flask containing 100 ml of sterile shake flask medium (glucose, 75g/l; soya meal, 20 g/l; MgSO₄.7H₂O, 2g/l; KH₂PO₄, 10g/l; K₂SO₄, 2g/l;

CaCl₂-2H₂O 0.5 g/l; Citric acid, 2g/l; yeast extract, 10g/l; trace metals [ZnSO₄.7H₂O, 14.3 g/l; CuSO₄.5H₂O, 2.5 g/l;

NiCl₂.6H₂O, 0.5 g/1; FeSO₄.7H₂O, 13.8 g/l, MnSO₄.H₂O, 8.5 g/l; citric acid, 3.0 g/l], 0.5 ml/l; urea, 2g/l, made with tap water and adjusted to pH 6.0 before autoclaving), and incubated at 37°C on a rotary shaker at 200 rpm for 18 hours. 50 ml of this culture is aseptically transferred to a 3 liter fermentor containing 1.8 liters of the fermentor media (maltodextrin MD01 300 g/l; MgSO₄-7H₂O, 2g/l; KH₂PO₄, 2g/l; citric acid 2g/l; K₂SO₄, 2.7 g/l;CaCl₂.2H₂O, 2g/l; trace metals, 0.5 ml/l; pluronic antifoam, lml/l; made with tap water and pH adjusted to 6.0 before autoclaving). The fermentor temperature is maintained at 34°C by the

circulation of cooling water through the fermentor jacket. Sterile air is sparged through the fermentor at a rate of 1.8 liter/min (lv/v/m). The agitation rate is maintained at 800 rpm for the first 24 hours after inoculation and at 1300 rpm for the remainder of the fermentation. The pH of the fermentation is kept at 4.0 by the automatic addition of 5N NaOH or H₃PO₄. Sterile feed (urea, 50 g/l; pluronic antifoam, 1.5 ml/l, made up with distilled water and autoclaved) is added to the fermentor by use of a peristaltic pump. The feed rate profile during the fermentation is as follows: 40 g of feed is added initially before inoculation; after inoculation, feed is at a constant rate of 2.5 g/l h.

Copper is made as a 400X stock in water or a suitable buffer, filter sterilized and added aseptically to the tank to a final level of 0.5 mM. Samples for enzyme activity determination are withdrawn and filtered through Miracloth to remove mycelia. These samples are assayed for laccase activity by a LACU assay. Laccase activity is found to increase continuously during the course of the fermentation, with a value of approximately 55 LACU/ml is achieved after 190 hours. This corresponds to approximately 350mg/l of recombinant laccase expressed.

IV. PURIFICATION OF RECOMBINANT LACCASE

MATERIALS AND METHODS

1. Materials.

Chemicals used as buffers and substrates are commercial products of at least reagent grade. Endo/N-glycosidase G is from Boehringer-Mannheim. Chromatography is performed on either a Pharmacia's FPLC or a conventional open column low pressure system. Spectroscopic assays are conducted on a Shimadzu PCI60 spectrophotometer.

2. Purification

(a) DSY2

2.8 liters cheese-cloth filtered broth (pH 7, 19mS) obtained from an A. oryzae pDSY2 transformant as described above is filtered on 0.45 μ Corning filter and concentrated on Spiral Concentrator (Amicon) with S1Y30 membrane to 200ml. The concentrate pH is adjusted to 7.5, diluted with 4.8 l water to achieve 1.2 mS, and concentrated on S1Y30 to 200ml. 50ml of this broth solution is applied onto a Q-Sepharose column(XKl6, 34ml gel), pre-equilibrated with 10mM Tris, pH 7.5, 0.7 mS (Buffer A). The blue laccase band that migrates slowly during loading is eluted by a linear gradient of Buffer B (Buffer A plus 0.5 M NaCl). 24 ml of pooled laccase fractions are concentrated on Centricon-100 (Amicon) to 4.5 ml and applied onto a Superdex 200 column (HiLoad 16/60, 120 ml gel). During the development with Buffer C (Buffer A plus 0.15 M NaCl, 14.4 mS), the blue laccase fractions elute followed by brownish contaminant fractions. Only the first half of the elution band(detected by Abs₆₀₀) show a high laccase to contaminant ratio and are pooled. The pooled fractions are dialyzed in lOmM Bis-Tris, pH 6.8,

0.6mS (Buffer D), applied onto a Mono-Q column (Mono-Q 5/5, 1ml) equilibrated with Buffer D, and eluted with Buffer E(Bufer D plus 0.5 M NaCl) using a linear gradient. The laccase fractions, which ome out round 27% Buffer E, are pure as judged by SDS-PAGE. At each step, the laccase fractions are routinely checked by ABTS oxidation, SDS-PAGE, and Western Blot.

(b) DSY10 2.8 liters cheese-cloth filtered broth(pH 7.3, 24mS) obtained from HowBl04-pDSY10 is filtered on Whatman #2 paper and concentrated on Spiral Concentrator (Amicon) with SlY100 membrane to 210ml. The concentrate pH is diluted with water to achieve 1.2 mS, and concentrated on SlY100 to 328 ml. This broth solution is applied onto a Q-Sepharose column(XK26, 120 ml gel), pre-equilibrated with 10mM Tris, pH 7.5, 0.7 mS(Buffer A). The blue laccase band that migrates slowly during loading is eluted by a linear

gradient of Buffer B (Buffer A plus 2 M NaCl). 120 ml of pooled laccase fractions are diluted with water to achieve 1.1mS and then concentrated on SIY100 to 294 ml and applied onto a Mono-Q column (HiLoad 16/10, 40 ml gel) pre- equilibrated with Buffer A. The laccase slowly passes through the column during loading and washing with Buffer A. The pooled fractions which have a pH reading of 5.6, are loaded on a Mono-Q column(HiLoad 16/10, 40 ml gel), pre- equilibrated with Buffer C(10mM MES, pH 5.5, 0.1 mS). The laccase fractions elute by a very shallow gradient of Buffer D (Buffer C + 1M NaCl). Enzymatic assays are conducted as described above.

3. Protein analysis

Total amino acid analysis, N-terminal sequencing, deglycosylation, SDS-PAGE, IEF, and Western blots are performed as decribed above.

B. RESULTS AND DISCUSSION

1. Purification and Characterization

Overall a 256-fold purification and a yield of 37% are achieved for DSY10, and a 246-fold purification and a yield of 14% are achieved for DSY2 In terms of electorphoretic pattern, spectral properties and activity, purified DSY2 and DSY10 are indistinguishable. Purified recombinant laccases behave as a dimer on gel filtration, and exhibit subunit molecular weight which is somewhat larger than that of the wild type laccase, indicating a post-translational processing in A. oryzae that results in the extra glycosylation on the recombinants. Deglycosylation has confirmed the difference in mass arising from extra sugars (Table 3).

The spectra of the purified laccases have maxima of 615 nm and 275, with the ratio of absorbance at 275 nm to that at 615 nm being 16, indicating one Type I Cu per subunit. The ratio of absorbance at 330nm to that at 615nm is 1.0, close to the 0.75 value of Rhus vernicefera laccase, suggesting the presence of one Type II and two Type III copper ions per subunit. The extinction coefficient determined by amino acid analysis is 1.71/(g*cm),

3. Activity

The laccase activity is measured by syringaldazine and ABTS oxidations. Expressed per A₂₇₅, the laccase has a value of 83 for LACU. Expressed per mg, it has a LACU of 141. The pH profile of the laccase is provided in Figure 9. V. USE OF POLYPORUS LACCASE TO DYE HAIR

The dyeing effect of Polyporus pinsitus laccase is tested and compared to the dyeing effect of 3% H₂O₂ on various dye precursors (listed below) and further on 0.1% p- phenylenediamine compared with a number of modifiers.

Materials:

Dye precursors:

0.1 % p-phenylene-diamine in 0.1 M K-phosphate buffer, pH

7.0. (pPD) 0.1 % p-toluylene-diamine in 0.1 M K-phosphate buffer, pH 7.0.

0.1 % chloro-p-phenylenediamine in 0.1 M K-phosphate buffer, pH 7.0.

0.1 % p-aminophenol in 0.1 M K-phosphate buffer, pH 7.0. 0.1 % o-aminophenol in 0.1 M K-phosphate buffer, pH 7.0. 0.1 % 3, 4-diaminotoluene in 0.1 M K-phosphate, buffer pH 7.0. Modifiers:

0.1 % m-phenylene-diamine in 0.1 M K-phosphate buffer, pH

7.0.

0.1 % 2, 4-diaminoanisole in 0,1 M K-phosphate buffer, pH

7.0.

0.1 % α-naphthol in 0.1 M K-phosphate buffer, pH 7.0.

0.1 % hydroquinone in 0.1 M K-phosphate buffer, pH 7.0.

0.1 % pyrocatechol in 0.1 M K-phosphate buffer, pH 7.0.

0.1% resorcinol in 0.1 M K-phosphate buffer, pH 7.0.

0.1 % 4-chlororesorcinol in 0.1 M K-phosphate buffer, pH 7.0.

When a modifier is used, the dye precursor p-phenylene- diamine is combined with one of the above indicated

modifiers so that the final concentration in the dyeing solution is 0.1 % with respect to precursor and 0.1 % with respect to modifier. The enzyme used is a recombinant laccase from Polyporus pinisitus, at a concentration of 10 LACU/ml . Other solutions used in the process are 3% H₂O₂ (in the final dye solution), and a commercial shampoo.

The quantitative color of the hair tresses is determined on a Datacolor Textflash 2000 (CIE-Lab) by the use of CIE-Lab parameters L* ("0"=black and "100"=white) combined with a* ("-"=green and "+"=red). DL* and Da* are the delta values of L* and a*, respectively, of a sample when compared to L* and a* of untreated hair. The Light fastness is determined under a day light bulb (D65) at 1000 LUX.

Hair tresses of blond European hair (1 gram) are used.

4 ml dye precursor solution (including modifier) is mixed with 1 ml laccase or 1 ml H₂O₂ on a Whirley mixer, applied to the hair tresses and kept at 30°C for 60 minutes. The hair tresses are then rinsed with running water, combed, and air dried.

The results of the dyeing effect test are displayed below in Table 4-6 and further in the graphs in Figures 10 to 12.

The oxidative hair dyeing is carried out as described above, except that 50 LACU/ml Polyporus pinsi tus laccase was used.

To test wash stability, the dyed hair tresses are wetted and washed for 15 seconds with 50 μl of commercial shampoo, and rinsed with water for 1 minute. The hair tresses are washed up to 20 times.

The results of the hair wash test are displayed in figure 13. It can be seen in figure 13 that the wash

stability of hair washed up to 20 times is excellent, when using Polyporus pinsitus laccase for oxidative dyeing.

To test light fastness, tresses of blond european hair are used for testing the light fastness of hair dyed using Polyporus pinsitus laccase in comparison to hair dyed using H₂O₂. p-phenylene-diamine is the dye precursor. The dyeing of the hair is carried out as described above. One hair tress is kept dark, while an other is kept at day light (i.e.

under a day light bulb (D65)), at approximately 1000 LUX) for up to 275 hours. The CIE-Lab-values are determined immediately after the dyeing of the hair, and further during exposure to day light.

The results of the test are displayed in figure 14. Figure 14 shows that the hair dyed with p-phenylene-diamine using Polyporus pinsitus laccase has the same light fastness as hair dyed using H₂O₂.

Deposit of Biological Materials

The following biological materials have been deposited under the terms of the Budapest Treaty with the Agricultural Research Service Patent Culture Collection, Northern

Regional Research Center, 1815 University Street, Peoria, Illinois, 61604 on May 25, 1994 and given the following accession numbers.

Deposit Accession Number

E. coli DH5α containing NRRL B-21263 pDSY22(41GEN; an -3.0 kb EcoRI insert)

E. coli DH5α containing NRRL B-21268

PDSY23 (41GEN; an -4.5 kb Mlul insert;

insert contains a small portion of the

EcoRI fragment of pDSY22 and sequences

5' to the EcoRI fragment)

E. coli XL-1 Blue containing NRRL B-21264

PDSY2K31GEN; an -7.7 kb EcoRI/BamHI

insert)

E. coli XL-1 Blue containing NRRL B-21265 pDSYl8(21GEN; an -8.0 kb BamHI insert)

E. coli DH5oc containing NRRL B-21266 pDSYl9(23GEN; an -4 kb Hindlll insert)

E. coli DH5α containing NRRL B-21267 pDSY20 (24GEN; an -8.5 kb EcoRI insert)

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Novo Nordisk Biotech, Inc.

(B) STREET: 1445 Drew Avenue

(C) CITY: Davis, California

(D) COUNTRY: United States of America

(E) POSTAL CODE (ZIP) : 95616-4880

(F) TELEPHONE: (916) 757-8100

(G) TELEFAX: (916) 758-0317

(i) APPLICANT:

(A) NAME: Novo Nordisk A/S

(B) STREET: NOVO Alle

(C) CITY: Bagsvard

(D) COUNTRY: Denmark

(E) POSTAL CODE (ZIP) : DK-2880

(F) TELEPHONE: +45.4444 8888

(G) TELEFAX: +45 4449 3256

(F) TELEX: 37304

(ii) TITLE OF INVENTION: PURIFIED POLYPORUS LACCASES AND

NUCLEIC ACIDS ENCODING SAME

(iii) NUMBER OF SEQUENCES: 10

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Novo Nordisk of North America, Inc.

(B) STREET: 405 Lexington Avenue, Suite 6400

(C) CITY and STATE: New York, New York

(D) COUNTRY: U.S.A.

(E) ZIP: 10174-6401

(v) 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 #1.25 (EPO)

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: Co be assigned

(B) FILING DATE: 15-June-1995

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 08/265,534

(B) FILING DATE: 24-June-1994

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Lowney, Karen A.

(B) REGISTRATION NUMBER: 31,274

(C) REFERENCE/DOCKET NUMBER: 4185.204-WO

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 212 867 0123

(B) TELEFAX: 212 878 9655

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2418 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE:

(A) ORGANISM: Polyporus pinsitus

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 414..464

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 534..589

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 710..764

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 879..934

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1001..1050

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1147..1197

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1354..1410

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1609..1662

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: join (413..465, 533..590, 709..765, 878..935,

1000..1051, 1146..1198, 1353..1411, 1608..1663)

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 520 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Polyporus pinsitus

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2880 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: intron (B) LOCATION: 544..592

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 837..899

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1014..1066

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1133..1187

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1284..1342

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1752..1815

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1873..1928

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 2136..2195

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: join(364..543 , 593..661, 716..835, 900..1013,

1067..1132, 1188..1283, 1343..1498, 1554..1751, 1816..1872, 1929..2135, 2196..2489)

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 662..715

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1499..1553

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 519 amino acids

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

(ii) MOLECULE TYPE: protein

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3102 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Polyporus pinsitus

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 666..720

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 790..845

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1125..1182

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1390..1450

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1607..1661

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1863..1918

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1976..2025

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 2227..2285

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 2403..2458

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 2576..2627

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: join (665..721, 789..846, 1124..1183, 1389..1451,

1606..1662, 1862..1919, 1975..2026, 2226..2286, 2402..2459, 2575..2628).

(2) INFORMATION FOR SEQ ID NO: 6

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 512 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS : single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Polyporus pinsitus

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2860 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 851..905

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1266..1320

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1351..1376

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1416..1468

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1625..1683

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1882..1934 (ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 2202..2252

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 2370..2425

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 2543..2599

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: join(540..725, 782..850, 906..1025, 1086..1265,

1321..1350, 1377..1415, 1469..1624, 1684..1881, 1935..2201, 2253..2369, 2426..2542, 2600..2653)

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 511 amino acids

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

(ii) MOLECULE TYPE: protein

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2925 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(Vi) ORIGINAL SOURCE:

(A) ORGANISM: Polyporus pinsitus

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 734..808

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 878..932

(ix) FEATURE:

(A) NAME/KEY: intron (B) LOCATION: 1051..1104

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1219..1270

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1336..1397

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1713..7744

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 2030..2085

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 2308..2375

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 2492..2569

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: join (733..809, 877..933, 1050..1105, 1218..1271, 1335..1398, 1712..1775, 2029..2086, 2307..2376, 2492..2570).

2542..2600).

(2) INFORMATION FOR SEQ ID NO: 10

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 527 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Polyporus pinsitus

Claims

What we claim is :

1. A DNA construct containing a sequence encoding a

Polyporus laccase.

2. The construct of Claim 1 which comprises a sequence encoding a Polyporus pinsitus laccase.

3. The construct of Claim 1 which comprises a nucleic acid sequence encoding the amino acid sequence depicted in SEQ ID

NO. 2.

4. The construct of Claim 1, which comprises the nucleic acid sequence depicted in SEQ ID NO. 1.

5. The construct of Claim 1 which comprises a nucleic acid sequence encoding the amino acid sequence depicted in SEQ ID NO. 4.

6. The construct of Claim 1, which comprises the nucleic acid sequence depicted in SEQ ID NO. 3.

7. The construct of Claim 1 which comprises a nucleic acid sequence encoding the amino acid sequence depicted in SEQ ID NO. 6.

8. The construct of Claim 1, which comprises the nucleic acid sequence depicted in SEQ ID NO. 5.

9. The construct of Claim 1 which comprises a nucleic acid sequence encoding the amino acid sequence depicted in SEQ ID NO. 8.

10. The construct of Claim 1, which comprises the nucleic acid sequence depicted in SEQ ID NO. 7.

11. The construct of Claim 1 which comprises a nucleic acid sequence encoding the amino acid sequence depicted in SEQ ID

NO. 10.

12. The construct of Claim 1, which comprises the nucleic acid sequence depicted in SEQ ID NO. 9.

13. The construct of Claim 1, which comprises the nucleic acid sequence selected from those contained in NRRL B-21263, 21264, 21265, 21266, 21267, and 21268.

14. A substantially pure Polyporus laccase enzyme.

15. The enzyme of Claim 14 which is a Polyporus pinsitus laccase.

16. The enzyme of Claim 14 which comprises the amino acid sequence selected from the group consisting of the sequences depicted in SEQ ID NOS. 4, 6, 8, and 10 or a sequence with at least about 80% homology thereto.

17. A recombinant vector comprising an DNA construct containing a sequence encoding a Polyporus laccase.

18. The vector of Claim 17 in which the construct is operably linked to a promoter sequence.

19. The vector of Claim 18 in which the promoter is a fungal or yeast promoter.

20. The vector of Claim 19 in which the promoter is the TAKA amylase promoter of Aspergillus oryzae.

21. The vector of Claim 18 in which the promoter is the glucoamylase (glaA) promoter of Aspergillus niger or

Aspergillus awamori .

22. The vector of Claim 17 which also comprises a selectable marker.

23. The vector of Claim 22 in which the selectable marker is selected from the group consisting of amdS, pyrG, argB, niaD, sC, trpC and hygB.

24. The vector of Claim 22 in which the selectable marker is the amdS marker of Aspergillus nidulans or Aspergillus oryzae, or the pyrG marker of Aspergillus nidulans,

Aspergillus niger, Aspergillus awamori , or Aspergillus oryzae.

25. The vector of Claim 18 which comprises both the TAKA amylase promoter of Aspergillus oryzae and the amdS or pyrG marker of Aspergillus nidulans or Aspergillus oryzae.

26. A recombinant host cell comprising a heterologous DNA construct containing a sequence encoding a Polyporus laccase.

27. The cell of Claim 26 which is a fungal cell.

28. The cell of Claim 27 which is an Aspergillus cell,

29. The cell of Claim 26 in which the construct is

integrated into the host cell genome.

30. The cell of Claim 26 in which the construct is

contained on a vector.

31. The cell of Claim 26 which comprises a construct containing a sequence encoding an amino acid sequence selected from the group consisting of those depicted in SEQ ID NOS. 2, 4, 6, 8, and 10.

32. A method for obtaining a laccase enzyme which comprises culturing a recombinant host cell comprising a DNA construct containing a nucleic acid sequence encoding a Polyporus laccase enzyme, under conditions conducive to expression of the enzyme, and recovering the enzyme from the culture.

33. A method for obtaining a laccase enzyme which comprises culturing a recombinant Aspergillus host cell comprising a DNA construct containing a nucleic acid sequence encoding a Polyporus-like laccase enzyme, under conditions conducive to expression of the enzyme, and recovering the enzyme from the culture.

34. A Polyporus enzyme obtained by the method of Claim 33.

35. A method for polymerizing a lignin or lignosulfate substrate in solution which comprises contacting the substrate with a Polyporus laccase.

36. A method for in situ depolymerization in Kraft pulp which comprises contacting the pulp with a Polyporus laccase.

37. A method for oxidizing dyes or dye precursors which comprises contacting the dye or dye precursor with a

Polyporus laccase.

38. A method for dyeing hair which comprises contacting a Polyporus laccase, in the presence or absence of at least one modifier, with at least one dye precursor, for a time and under conditions sufficient to permit oxidation of the dye precursor to a dye.

39. The method of claim 38 in which the dye precursor is selected from the group consisting of a diamine,

aminophenol, and a phenol.

40. The method of claim 38, wherein the modifier, when used, is a meta-diamine, a meta-aminophenol or a polyphenol.

41. The method of claim 38 in which the dye precursor is a primary intermediate selected from the group consisting of an ortho- or para-diamine or aminophenol.

42. The method of claim 38 in which more than one dye precursor is used.

43. The method of claim 38 in which more than one modifier is used.

44. The method of claim 38 in which both a primary

intermediate and a modifier are used.

45. A dye composition comprising a Polyporus laccase combined with at least one dye precursor.

46. A dye composition comprising a Polyporus laccase combined with at least one primary intermediate and at least one modifier.

47. A container containing a dye composition comprising a Polyporus laccase and at least one dye precursor in an oxygen-free atmosphere.

48. The container of claim 47 which contains at least one primary intermediate dye precusor combined with at least one modifier.

49. A method of polymerizing or oxidizing a phenolic or aniline compound which comprises contacting the phenolic or aniline compound with a Polyporus laccase.