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1. WO2011000121 - POLYPEPTIDES HAVING TYROSINASE ACTIVITY AND USES THEREOF

Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

[ EN ]

Polypeptides having tyrosinase activity and uses thereof

Technical Field

The present invention relates to enzymes, in particular to tyrosinases, useful in the biochemical synthesis of melanins.

Background Art

Melanins are a group of diverse homo- and heteropolymers principally formed of aromatic units. Depending on the chemical precursors, different types of melanins exist, each having different physical chemical properties. The different physical chemical properties make them attractive for use in different industrial applications: currently, melanins are used in medical and cosmetic products and treatments, and also for the elimination of metals in the treatment of water.

Melanins are the principal pigment present in the surface of animals, but are also synthesized by bacteria and fungi. Their biotechnological synthesis is an interesting alternative to the chemical synthesis, as the latter often produces a highly variable product and the production costs are high. Biotechnological synthesis of melanin involves the oxidation of phenolic compounds.

This process is called melanogenesis and depends on the action of specific enzymes belonging to the family of mo-nophenoloxidases, in particular tyrosinases (EC

1.14.18.1) .

Tyrosinases are copper containing enzymes found throughout the kingdoms of life, for an overview see Mayer (2006), Phytochem., Vol. 67, pp. 2318-2331; Claus and Decker (2006), System. Appl. Microbiol., Vol. 29, pp. 3-14) . These enzymes have as principal substrate the amino acid L-tyrosine which is converted to L-DOPA and in a subsequent step to dopaquinone which in turn is a precursor in melanin biosynthesis. A particular charac- teristic of tyrosinases is that they have two types of activities: they are monophenoloxidases and o-diphenoloxidases. Hence, tyrosinases can hydroxylate mo-nophenols and convert them into o-diphenols; they also can oxidize o-diphenols converting them into o-quinones (see fig. 1) .

Most intensively studied examples are tyrosinases from Streptomyces sp. and fungal species. In the case of the microorganism Streptomyces, the protein MeICl was identified as a copper chaperone, transferring copper to the apotyrosinase, thus activating the tyrosinase MelC2. The genes that codes for these two proteins form an operon in the genome of Streptomyces.

Although tyrosinases of streptomycetes are widely used, they have a number of disadvantages: for achieving a high expression level, the copper chaperone needs to be expressed simultaneously with the tyrosinase. For the producing microorganism, e.g. a recombinant E. coli, this represents a high metabolic load, which delim-its the yield of the process. Moreover, codon usage in E. coli differs to Streptomyces, which leads to deficient protein expression.

An example of a tyrosinase being independent of a copper chaperone is described in MXPA04004786 which discloses a microbial tyrosinase stemming from Rhizobium etli CFN42. A mutant thereof having the mutation D535G showed enhanced enzyme activity.

However, commercially available tyrosinases are typically crude preparations having multiple impuri-ties which is inconvenient for chemical synthesis. As these preparations generally stem from mushrooms, the enzyme is often not uniform due to the presence of isoenzymes and different glycolsylation patterns. Moreover, the production from a natural source results usually in high batch to batch variations.

Therefore, there is an urgent need for tyrosinases which can be produced by high level expression and can easily be purified.

Disclosure of the Invention

Hence, it is a general object of the invention to provide a tyrosinase which can be easily produced in a recombinant manner and at high yields.

Therefore, in a first aspect, the invention provides an isolated tyrosinase of Verrucomicrobium spinosum and mutants thereof having enhanced activity.

Furthermore, it has surprisingly been found that a fragment of the V. spinosum tyrosinase has a 30-50 fold increased activity when compared to the entire se-quence (the so-called pro-form, pro-tyrosinase) . The fragment may e.g. be produced by proteolytic processing or by recombinant expression of SEQ ID No. 7 or a variant thereof.

In further aspects, the present invention provides methods for the production of melanin, for the crosslinking of proteins as well as for the cross-linking of phenols.

Brief Description of the Drawings The invention will be better understood and objects other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such description makes reference to the annexed drawings, wherein:

Figure 1 shows exemplary monophenolase activity (Fig. Ia) and diphenolase activity (Fig. Ib) of a tyrosinase.

Figure 2 shows the annotated DNA and amino acid sequence of pre-pro-tyrosinase.

Figure 3 shows the structure of the pre-pro-tyrosinase protein. 1: TAT signal peptide, amino acids 1- 36; 2: mature domain, amino acids 37-357; 3: C-terminal extension amino acids 358-518; 4: copper binding motifs.

Figures 4 a and b show the activity of the pro-tyrosinase at different pH and temperatures, respec-tively.

Figure 5 shows a Coomassie- stained SDS poly-acrylamide gel. Lane 1 and 8: Molecular weight markers. Lane 2: Pro-tyrosinase control 24 hours at room temperature. Lane 3: Trypsin contol 24 hours at room tempera-ture. Lane 4: Pro-tyrosinase plus trypsin 0 hours at room temperature. Lane 5: Pro-tyrosinase plus trypsin 1 hour at room temperature. Lane 6: Pro-tyrosinase plus trypsin 4 hours at room temperature. Lane 7: Pro-tyrosinase plus trypsin 24 hours at room temperature.

Figure 6 shows the enzymatic activity of the pro-tyrosinase and the trypsin digested form of the pro-tyrosinase.

Figure 7 A-H depict SDS PAGE gels showing the cross-linking of various model protein substrates by recombinant V. spinosum tyrosinase. In all gels M = Markers with weight in kDa indicated; 1 = Protein substrate; 2 = Protein substrate + tyrosinase; 3 = Protein substrate + caffeic acid; 4 = Protein substrate + caffeic acid + tyrosinase; 5 = Protein substrate + phenol; 6 = Protein substrate + phenol + tyrosinase. In all cases the final concentrations are: protein substrate lmg/mL, tyrosinase 0. lmg/mL and caffeic acid or phenol (when present) 0.1 mg/mL. In gel A are shown the results obtained with bovine casein sodium salt (BNaC) , in gel B with bovine α-casein (BaC) , in gel C with bovine β-casein (BβC) , in gel D with bovine κ-casein (BK) ; in gel E with horse myoglobin (HMb) ; in gel F with horse heart cytochrome C (HHC) ; in gel G with hen egg white lysozyme (HEWL) ; in gel E with Candida antartica lipase B CALB. The position of V. spinosum tyrosinase is indicated in all gels.

Figure 8 depicts the effect of various parameters on the yield of activity found in CALB-phenol- tyrosinase aggregates. In charts A-C the cross-hatched bars show the lipase activity in the initial reaction mixture; the bars with horizontal stripes the activity in the supernatant of the clarified reaction mixture; the white bars with black dots the activity in the pellet of the clarified reaction mixture; the bars with angled stripes the activity retained in the pellet after washing 5 times. Chart A shows the effect of phenol concentration (0-40 mg/mL) on the activities obtained; Chart B the ef-feet of tyrosinase concentration (0-0.4 mg/mL) on the activities; Chart C the effect of CALB concentration (0-4 mg/mL) on the activities obtained. All incubations were done in 0. IM sodium phosphate buffer pH7 and when not varied the phenol concentration was kept constant at 30 mg/mL, the tyrosinase concentration at 0.04 mg/mL and the CALB concentration at 2 mg/mL. Chart D shows the effect of an overnight incubation at various temperatures and pHs on activity loss. White bars with black dots and chequered bars show the activities present in control and immobilised CALB respectively incubated at the indicated pH at 300C overnight (18h) ; Bars with horizontal and bars with angled stripes show the activities present in control and immobilised CALB respectively incubated at the indicated pH at 45°C overnight; Cross-hatched bars and bars with vertical stripes show the activities present in control and immobilised CALB respectively incubated at the indicated pH at 600C overnight. Shown is the average of three independent experiments with the error as indicated.

Modes for Carrying Out the Invention

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present in- vention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Various aspects of the invention are described in further detail in the following subsections. It is understood that the various embodiments, preferences and ranges may be combined at will. Further, de-pending on the specific embodiment, selected definitions, embodiments or ranges may not apply.

The polypeptide of the present invention can be isolated from V. spinosum, preferably from V. spinosum DSM 4136. V. spinosum is a Gram-negative prosthecate bac-terium originally isolated from a fresh water lake. The strain was deposited at the Deutsche Stammsammlung fur Mikroorganismen under accession No. DSM 4136 and is also available as ATCC 43997 or IFAM 1439. The genome of V. spinosum has been sequenced and whole genome shotgun se-quences have been submitted to public databases and may e.g. be accessed via The Institute for Genomic Research (TIGR project ID number 10620) or through PudMed, see access no. NZ_ABIZ00000000.

Annotation of the V. spinosum tyrosinase gene (Locus Tag on the V. spinosum genome

VspiD_010100001190) was added automatically, according to which the gene sequence encodes a polypeptide consisting of a TAT signal peptide (amino acids 1-36; SEQ ID No. 4) at its N-terminal end, the mature domain (amino acids 37-357; SEQ ID No. 3) and a C-terminal extension (amino acids 358-518; SEQ ID No. 5). However, up to now, the tyrosinase enzyme has not been isolated or produced in recombinant manner.

As used herein, the "pro-form" or "pro-tyrosinase" refers to the V. spinosum tyrosinase having SEQ ID No. 1, comprising the catalytic domain (amino acids 37-357; SEQ ID No. 3) and a C-terminal extension (amino acids 358-518; SEQ ID No. 5), but lacking the TAT signal peptide (amino acids 1-36; SEQ ID No. 4). In contrast, the pre-pro-tyrosinase (SEQ ID No. 2), comprises the catalytic domain (amino acids 37-357; SEQ ID No. 3), a C-terminal extension (amino acids 358-518; SEQ ID No. 5) and the TAT signal peptide (amino acids 1-36; SEQ ID No. 4) .

It has surprisingly been found that a mutant (SEQ ID No. 6) of the pro-form carrying alanine in the C-terminal extension, in particular at position 453 of SEQ ID No. 2 or in Fig. 2 labelled as gate keeper, led to an increased enzymatic activity when compared to the pro-form. In particular, the Kcat °f said mutant is approximately Is"1 using ImM L-tyrosine and lOμM L-DOPA as sub-strates. Thus, in one aspect, the invention provides a polypeptide having tyrosinase activity and also (i) having a molecular weight of approximately 54 kDa; (ii) having a pi of approximately 6,9, (iii) having a Kca^_ of approximately Is"1; and/or (v) having at least 50%, prefera-bly at least 55%, 60%, 65%, 70%, 75% 80%, 85%, 90%, such as at least 95%, for instance 96%, 97%, 98%, 99% or 100%, identity to SEQ ID No. 6 with the proviso that the amino acid at position 453 is not phenylalanine, i.e. the naturally occurring amino acid at said position. It is to be understood that said sequence identity is given over at least the first 350 consecutive amino acids, more preferably over at least the first 400 consecutive amino acids, even more preferably over at least the first 450 consecutive amino acids and most preferably over the en-tire length of SEQ ID No. 6.

^cat as used herein refers to the catalytic constant. Preferably, Kca^ is determined using ImM L-Tyrosine, lOuM L-DOPA and 0,1M Potassium Phosphate pH7 at 300C.

The polypeptide lacks part of or preferably the entire TAT signal peptide (SEQ ID NO. 4) . The TAT signal peptide as used herein refers to amino acids 1-36 of the native V. spinosum tyrosinase. The signal peptide of SEQ ID NO. 4 may be replaced by another signal peptide or it may be totally lacking.

Further variants may comprise deletions, preferably at the C-terminal end, with the proviso that no phenylalanine, preferably neither phenylalanine nor tyrosine nor leucine is present at position 453. An exemplary amino acid that at position 453 enhances the activity in comparison to the entire sequence comprising phenylalanine at position 453 is alanine. Thus, in a preferred embodiment, the invention provides a polypeptide having at least 50%, preferably at least 55%, 60%, 65%, 70%, 75% 80%, 85%, 90%, such as at least 95%, for instance 96%, 97%, 98%, 99% or 100%, identity to SEQ ID No. 6 with the proviso that the amino acid at position 453 is alanine. It is to be understood that said sequence identity is given over at least the first 350 consecutive amino acids, more preferably over at least the first 400 consecutive amino acids, even more preferably over at least the first 450 amino consecutive acids and most preferably over the entire length of SEQ ID No. 6.

In one embodiment, variants of the polypeptides are provided, comprising one or more amino acid residues modified by amino acid substitution, addition, deletion or insertion. In a much preferred embodiment, the polypeptide comprises at its N-terminal end the motif MA (i.e. methionine and alanine at amino acid positions 1 and 2, respectively) .

The inventors have also surprisingly found that a fragment of the pro-form of V. spinosum tyrosinase has an activity being 30-50 fold higher than the pro-form. Thus, in another aspect, the invention provides a polypeptide comprising a fragment on the V. spinosum tyrosinase or a derivative thereof, wherein said fragment comprises the mature domain of the V. spinosum tyrosinase or a functional fragment of said mature domain.

The mature domain, as used herein, refers to amino acids 37-357 (SEQ ID No. 3) which has tyrosinase activity.

The term "functional fragment" refers to a fragment being shorter than the SEQ ID No. 3, i.e. having deletions, but still having tyrosinase activity.

In comparison to the pro-form, preferably parts of the C-terminal extension, more preferably the entire C-terminal extension is lacking. The C-terminal extension as used herein refers to amino acids 358-518 of the native V. spinosum tyrosinase (SEQ ID No. 5) . Additionally or alternatively, parts of the TAT signal peptide (SEQ ID No. 4), more preferably the entire signal peptide is lacking. In a preferred embodiment, the poly-peptide comprises at its N-terminal end the motif MA (e.g. SEQ ID No. 7) .

Therefore, in a preferred embodiment, the present invention provides a polypeptide being a fragment of the pro-form of the V. spinosum tyrosinase, wherein the polypeptide consists of amino acids 37-357 of the pro-form (i.e. the mature V. spinosum tyrosinase, see SEQ ID No. 3) or a functional fragment thereof.

In one embodiment, a polypeptide of the present invention consists of or comprises a sequence based on the mature V. spinosum tyrosinase (i.e. the mature domain) carrying mutations in specific amino acid residues and/or having additional amino acids at the N-terminal or at the C-terminal end.

In one embodiment, variants of the polypep-tides are provided. A "variant" as used herein is either naturally occurring variation of a given polypeptide or may be a recombinantIy prepared or otherwise modified variation of a given peptide or protein in which one or more amino acid residues have been modified by amino acid substitution, addition, deletion or insertion. Hence, in a preferred embodiment, the invention provides an isolated polypeptide comprising an amino acid sequence hav- ing at least 50%, preferably at least 55%, 60%, 65%, 70%, 75% 80%, 85%, 90%, such as at least 95%, for instance 96%, 97%, 98%, 99% or 100% identity, to the amino acid sequence in SEQ ID No. 2, to SEQ ID No. 7 or to SEQ ID No. 5 with the proviso that the amino acid at position 453 is not phenylalanine. It is to be understood that said sequence identity is given over at least 350 consecutive amino acids, more preferably over at least 400 consecutive amino acids, even more preferably over at least 450 consecutive amino acids or over at least 500 consecutive amino acids and most preferably over the entire length of SEQ ID No. 2. In case of SEQ ID No. 5, said sequence identity is given over at least 220 consecutive amino acids, more preferably over at least 300 consecutive amino acids and most preferably over the entire length of SEQ ID No. 5. In case of SEQ ID No. 7, said sequence identity is given over at least 200 consecutive amino acids, more preferably over at least 250 consecutive amino acids, even more preferably over at least 300 consecutive amino acids and most preferably over the entire length of SEQ ID No. 7.

In a much preferred embodiment, the amino acid at position 453 of SEQ ID No. 5 is alanine. Preferably, the variant has tyrosinase activity, preferably a κcat of at least Is"1 using ImM L-tyrosine and lOμM L-DOPA as substrates.

As used herein, "identity" refers to the sequence matching between two polypeptides, molecules or between two nucleic acids. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, then the respective molecules are identical at that position. The "percentage identity" between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps or inserts, and the length of each gap or insert, which need to be introduced for optimal alignment of the two sequences. The length of gaps or inserts in the meaning of the present invention in general does not exceed about 15 amino acids. Generally, a comparison is made when one or more sequences of the present invention are aligned with one or more query se-quences to give maximum identity. Such alignment can be provided using, for instance, the method of the Needleman and Wunsch (J. MoI. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. When indicated that a certain sequence identity is given over at least N consecutive amino acid or nucleotides, respectively, then it is meant that the query sequence has at least that number N of amino acids or nucleotides, respectively.

In one embodiment, the polypeptide comprises additional amino acids at the N-terminal end of SEQ ID No. 3, such as 1, 2, 3, 4, or up to 10 amino acids of the TAT signal peptide (SEQ ID NO. 4). In a much preferred embodiment, a MA-motif is present at the N-terminal end (SEQ ID NO. 7) . Thus, in one embodiment, the polypeptide is or comprises SEQ ID No. 7.

Additionally or alternatively, the polypep-tide comprises at the N-terminal end of SEQ ID No. 3 or SEQ ID No. 7 a signal peptide other than the TAT signal peptide to direct a protein of the present invention into the secretory pathway of the host cells. The choice of the signal peptide is often linked to the choice of the host cell. Non-limiting examples include PeIB, OmpA, DsbA, PhoA, SacB for expression in B. subtilits or α-factor signal peptides for expression in P. pastoris.

Additionally or alternatively, the polypeptide may comprise a N-terminal tag or a C-terminal tag, e.g. a C-tag, i.e. a stretch of amino acids added to the N- or the C-terminal end of SEQ ID No. 3 or SEQ ID No. 7. This may e.g. be 1, 2, 3, 4, 5, 6 or more preferably up to 20, 40, 50, or 100 amino acids of the N- or the C-terminal extension of the V. spinosum tyrosinase, with the provisio that not the entire C-terminal extension is present. In case amino acid 453 is present, it is pref-erably substituted by alanine.

In this embodiment, the polypeptide has preferably a Kcat twice as low as the mature form for a x:y mixmixture of ImM L-tyrosine and 10μM L-DOPA.

Additionally or alternatively, the polypep-tides of the present invention may comprise a C-tag, e.g. to simplify the purification of the polypeptide or to allow for a visible read-out. Non limiting examples include a His-tag, GST-tag, FLAG-tag, HA-tag or a MYC-tag. In a preferred embodiment, the tag can be recognized by an an-tibody. In a much preferred embodiment, the polypeptide comprises a C-terminal tag different to SEQ ID No. 5, i.e. to the native C-terminal extension of V. spinosum tyrosinase.

In one embodiment, the polypeptide comprises a chemical tag, such as biotin. Preferably, said chemical tag is present at the C-terminus.

The polypeptides of the present invention may be produced by (i) genetic engineering and recombinant expression or (ii) by proteolytic processing of the na-tive pro-tyrosinase of V. spinosum, preferably from V. spinosum DSM 4136, wherein the pro-form is digested by a protease, e.g. trypsin. Preferably, said protease cleaves after the Y-N-Y motif present at amino acids 347 to 349.

In one embodiment, the present invention thus provides a polypeptide which can be isolated from V.

spinosum or recombinantIy expressed, (i) having tyrosinase activity, (ii) having a molecular weight of approximately 37 kDa; (iii) having a pi of approximately 7,1; (iv) having a Kcat of approximately Is"1; and/or (v) hav-ing at least 50%, preferably, 55%, 60%, 65%, 70%, 75%,

80%, 85%, 90%, 95%, more preferably 96%, 97%, 98%, 99% or 100% identity to SEQ ID No. 3. It is to be understood that said sequence identity is given over at least the first 220 consecutive amino acids, even more preferably over at least the first 300 consecutive amino acids and most preferably over the entire length of SEQ ID No. 3. More preferably, when using a mixture of ImM L-tyrosine and 10μM L-DOPA, the enzyme shows a pH optimum of ca 6,5 and a temperature maximum at 450C.

The mature domain comprises two copper binding motifs. However, the protein is independent of a co-expressed copper-chaperone. Therefore, in one embodiment, the polypeptides of the present invention are expressed in absence of copper and can be reconstituted with copper upon lysis of the host cells. After purification, the so reconstituted polypeptide shows absorbance at 340nm which is a characteristic of copper binding proteins. Moreover, atomic absorbance spectroscopy shows about 2 copper atoms per active purified protein molecule.

Nucleic acids, vectors, expression systems In a further aspect, the present invention provides an isolated nucleic acid molecule encoding the polypeptide disclosed herein. The term "nucleic acid molecule," as used herein refers to DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.

In a preferred embodiment, the isolated nucleic acid has a sequence comprised in SEQ ID No. 9 or at least 50%, preferably at least 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. It is to be understood that said sequence identity is given over at least 800 consecutive nucleotides, more preferably over at least 850 consecutive nucleotides, even more preferably over at least 900 con-secutive nucleotides and most preferably over the entire length of SEQ ID No. 9.

Preferably, the isolated nucleic acid sequence encodes a peptide having tyrosinase activity.

Optionally, the nucleic acid may further encode a polypeptide sequence adjacent to the mature domain of the tyrosinase, other than SEQ ID No. 4 and/or SEQ ID No. 5, such as an N-terminal signal sequence and/or a C-terminal His-tag.

Preferably, said isolated nucleic acid comprises SEQ ID No. 8 or a fragment thereof, with the pro-viso that said isolated nucleic acid has not the sequence of the naturally occurring pre-pro-tyrosinase (see fig. 2) . For example, the entire or partial TAT signal peptide may be lacking and/or the encoded amino acid at position 453 of SEQ ID No. 5 -if present- is not phenylalanine and/or the sequence encodes at a given position for an amino acid different to the one indicated in Fig. 2.

In one embodiment, the present invention provides a recombinant vector comprising a nucleic acid construct according to the present invention.

The recombinant vector into which the nucleic acid construct of the invention is inserted may be any vector. Often, the choice of vector depends on the host cell into which it is to be introduced. 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. Alternatively, the vector may be one which integrates into the host cell genome and replicates together with the chromosome (s) into which it has been integrated.

In a preferred embodiment, the vector is an expression vector in which the DNA sequence encoding the polypeptide of the invention is operably linked to additional segments required for transcription of the DNA. The term "operably linked" indicates that the nucleic acid encoding the polypeptide is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the 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. Moreover, the promoter may be inducible or constitutive.

Examples of suitable promoters for use in bacterial host cells include the Escherichia coli lac, trp or tac promoters or Bacillus subtilis p43, spaC or xylA promotors. Suitable promoters for use in yeast host cells, preferably in Pichia pastoris, include GAP or AOX promoters. Suitable promoters for use in filamentous fungus host cells, preferably Aspergillus sp., include amyA or adhA promoters.

The DNA sequence encoding the protein of the invention may also, if necessary, be operably connected to a suitable terminator, such as the human growth hormone terminator or (for fungal hosts) the TPM or ADH3 terminators. In particular in the case of eukaryotic host cells, the vector may further comprise elements such as polyadenylation signals (e.g. from SV40 or the adenovirus 5 EIb region), transcriptional enhancer sequences (e.g. the SV40 enhancer) and translational enhancer sequences (e.g. the ones encoding adenovirus VA RNAs).

The recombinant vector of the invention may further comprise an origin of replication, i.e. a DNA sequence enabling the vector to replicate in the host cell in question.

The vector may further comprise one or more selection markers, e.g. one which confers resistance to a drug (non-limiting examples inlcude ampicillin, kanamy-cin, tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate) ; or a gene the product of which complements a defect in the host cell. For fungi, selectable markers include ade2, ura3, amdS or pyrG .

In humans, tyrosinases are involved in disease states such as melanoma, albinism and vitiligo.

Therefore, the vector disclosed herein could be used in gene therapy approaches.

The invention further encompasses a host cell comprising the vector and/or the nucleic acid disclosed herein. A variety of host cells are suitable for the expression of the polypeptides disclosed herein. For example, the polypeptides can be actively expressed in the E. coli cytoplasm. Further preferred host cells, without being limited to, include B. subtilis and P. pastoris.

In one aspect, the invention provides a method for the production of a polypeptide disclosed herein, comprising the steps of

(a) providing a host cell comprising a vector and/or a nucleic acid encoding a polypeptide disclosed herein;

(b) cultivating the host cell under conditions such that the polypeptide is expressed; and

(c) optionally purifying the polypeptide.

Industrial Application of the polypeptides disclosed herein

Melanin has many applications as it absorbs radiation (UV, X-ray, gamma) . Therefore, melanin may for example, without being limited to, be used as a antioxi-dant, drug carrier or cation exchanger. As tyrosinases are involved in melanin synthesis, they can for example be used to manufacture powerful antioxidants, e.g. the antioxidant and anticarcinogenic caffeic acid and the antioxidant hydroxytyrosol. Thus, in one aspect, the inven-tion provides a method for the production of melanin comprising the steps of

(a) providing L-tyrosine

(b) providing the tyrosinase disclosed herein; and

(c) reacting the L-tyrosine with the tyrosinase under oxidizing conditions.

In another aspect, the invention provides a method for the cross-linking of phenols comprising the steps of

(a) providing mono- and/or bisphenols;

(b) providing one or more polymers having amino-, SH-, imidazol- and/or phenol groups as side chains;

(c) providing the tyrosinase disclosed herein; and

(d) reacting the phenols with the polymers and the tyrosinase under oxidizing conditions.

Being active towards environmental contaminants such as phenols, chlorophenols and pentachlorophe-nol, tyrosinases may e.g. be used in bioremediation pro-cedures .

A further application of tyrosinases is the formation of covalent cross-links between amino acid residues in proteins or proteinaceous material. Tyrosinase may form inter- and intramolecular cross-links be-tween tyrosine residues which results in polymerization. Such modification by cross-linking is frequently used e.g. in food processing. Regarding food quality, texture is a very essential factor which is not only related to sensory perception but also to water holding capacity, gelling and emulsifying properties and stability. Applications of tyrosinases in cross-linking food proteins have e.g. been described in WO06/084953, WO07/141385, EP0947142 and WO07/093674. However, also in other areas the cross-linking of proteins is used, e.g. wool fibers (WO02/14484) . Thus, in a preferred embodiment, the polypeptide of the present invention is used to cross-link a food protein, a proteinaceous fiber or a fiber-derived polymer, and, in another aspect, the invention provides a method for cross-linking a protein, which comprises re-acting a multi-copper oxidase with a protein under oxidizing conditions, thereby effecting cross-linking of the protein. Thereby, higher molecular weight forms, dimers, trimers and aggregates can e.g. be formed. The cross-linking activity is enhanced by adding phenols such as tyrosine or caffeic acid to the reaction.

The method for the cross-linking of proteins comprises the steps of

(a) providing a first polypeptide comprising tyrosine at one or more positions;

(b) optionally providing a second polypeptide comprising tyrosine at one or more positions;

(c) providing the tyrosinase disclosed herein; and

(d) reacting the first and optionally the second polypeptide with the tyrosinase under oxidizing conditions .

Preferably, tyrosine, caffeic acid or another suitable enhancing phenolic is added to the reaction-, more preferably to any one of steps a, b and c.

The sequences of the present invention are the following:

SEQ ID No. 1: pro-tyrosinase

KYHRLNLQNPAAAPFLESYKKAITVMLQLPPSDARNWYRNAFIHT LDCPHGNWWFWWHRGYTGWFERTVRELSGDPNFAFPYWDWTALPQVPDSFFNGVLD PNNPAFIASYNEFYSQLSNPMSALWNSFSTAQLQQMRNRGFQSVNDVWQAVRDSPMF FPRGRARTLTRQNPGFDATTRRAVSIGTIRNALAPTDFITFGSGKTANHSESATQGI LESQPHNNVHNNIGGFMQDLLSPTDPVFFAHHSNIDRLWDVWTRKQQRLGLPTLPTG ANLPLWANEPFLFFIGPDGKPVAKNKAGDYATIGDFDYNYEPGSGEAVIPAASRPGE MNNKVWLGTLGAAVPNFSASARADVMVPEAVPEAAMKADGPAVFAKITIAPPMDVAG VEFHVLVNPPENVSHVDFDSPSFAGTFSVFGKQLGGHKNQPLSFLMPLTEAVKKLQE TNELKPGQPLRVQWAERKGVNLTPLQAKVSEISVGTF

SEQ ID No. 2: pre-pro-tyrosinase

MSPPTTSRRQFLVTAGAAAASAGWSFGQEPAQAATAKYHRLNLQN PAAAPFLESYKKAITVMLQLPPSDARNWYRNAFIHTLDCPHGNWWFWWHRGYTGWF

ERTVRELSGDPNFAFPYWDWTALPQVPDSFFNGVLDPNNPAFIASYNEFYSQLSNPM

SALWNSFSTAQLQQMRNRGFQSVNDVWQAVRDSPMFFPRGRARTLTRQNPGFDATTR RAVSIGTIRNALAPTDFITFGSGKTANHSESATQGILESQPHNNVHNNIGGFMQDLL SPTDPVFFAHHSNIDRLWDVWTRKQQRLGLPTLPTGANLPLWANEPFLFFIGPDGKP VAKNKAGDYATIGDFDYNYEPGSGEAVIPAASRPGEMNNKVWLGTLGAAVPNFSASA RADVMVPEAVPEAAMKADGPAVFAKITIAPPMDVAGVEFHVLVNPPENVSHVDFDSP SFAGTFSVFGKQLGGHKNQPLSFLMPLTEAVKKLQETNELKPGQPLRVQVVAERKGV NLTPLQAKVSEISVGTF

SEQ ID No . 3 : mature domain

YHRLNLQNPAAAPFLESYKKAITVMLQLPPSDARNWYRNAFIHTL DCPHGNWWFWWHRGYTGWFERTVRELSGDPNFAFPYWDWTALPQVPDSFFNGVLDP NNPAFIASYNEFYSQLSNPMSALWNSFSTAQLQQMRNRGFQSVNDVWQAVRDSPMFF PRGRARTLTRQNPGFDATTRRAVSIGTIRNALAPTDFITFGSGKTANHSESATQGIL ESQPHNNVHNNIGGFMQDLLSPTDPVFFAHHSNIDRLWDVWTRKQQRLGLPTLPTGA NLPLWANEPFLFFIGPDGKPVAKNKAGDYATIGDFDYNYEPGSGEAV

SEQ ID No. 4: TAT signal peptide MSPPTTSRRQFLVTAGAAAASAGWSFGQEPAQAATA

SEQ ID No. 5: C-terminal extension

IPAASRPGEMNNKVWLGTLGAAVPNFSASARADVMVPEAVPEAAM KADGPAVFAKITIAPPMDVAGVEFHVLVNPPENVSHVDFDSPSFAGTFSVFGKQLGG HKNQPLSFLMPLTEAVKKLQETNELKPGQPLRVQWAERKGVNLTPLQAKVSEISVG TF

SEQ I D No . 6 : pro-tyrosinase having alanine at pos ition 453

YHRLNLQNPAAAPFLESYKKAITVMLQLPPSDARNWYRNAFIHTL DCPHGNWWFWWHRGYTGWFERTVRELSGDPNFAFPYWDWTALPQVPDSFFNGVLDP NNPAFIASYNEFYSQLSNPMSALWNSFSTAQLQQMRNRGFQSVNDVWQAVRDSPMFF PRGRARTLTRQNPGFDATTRRAVSIGTIRNALAPTDFITFGSGKTANHSESATQGIL ESQPHNNVHNNIGGFMQDLLSPTDPVFFAHHSNI DRLWDVWTRKQQRLGLPTLPTGA

NLPLWANEPFLFFIGPDGKPVAKNKAGDYATIGDFDYNYEPGSGEAVIPAASRPGEM NNKVWLGTLGAAVPNFSASARADVMVPEAVPEAAMKADGPAVFAKITIAPPMDVAGV EFHVLVNPPENVSHVDFDSPSFAGTFSVAGKQLGGHKNQPLSFLMPLTEAVKKLQET NELKPGQPLRVQWAERKGVNLTPLQAKVSEISVGTF

SEQ ID NO. 7: recombinant fragment comprising mature tyrosinase

MAKYHRLNLQNPAAAPFLESYKKAITVMLQLPPSDARNWYRNAFI HTLDCPHGNWWFVVWHRGYTGWFERTVRELSGDPNFAFPYWDWTALPQVPDSFFNGV LDPNNPAFIASYNEFYSQLSNPMSALWNSFSTAQLQQMRNRGFQSVNDVWQAVRDSP MFFPRGRARTLTRQNPGFDATTRRAVSIGTIRNALAPTDFITFGSGKTANHSESATQ GILESQPHNNVHNNIGGFMQDLLSPTDPVFFAHHSNIDRLWDVWTRKQQRLGLPTLP TGANLPLWANEPFLFFIGPDGKPVAKNKAGDYATIGDFDYNYEPGSGEAV

SEQ ID NO. 8: DNA sequence of recombinant pro-tyrosinase

gcaaa ataccaccgg ctcaatttac aaaaccctgc ggctgcacct ttcctggaga gctacaagaa ggccatcacc gtcatgctgc agctcccgcc ctcggatgcg cgcaactggt accggaacgc cttcatccac acgctggact gcccgcatgg gaactggtgg tttgtcgtgt ggcaccgtgg ctacaccggc tggtttgagc gcacggtccg cgagctcagt ggggacccca acttcgcctt tccttattgg gactggacgg cgctgccgca ggttccggac tctttcttca atggtgtgct ggaccccaac aatcctgcct tcattgccag ctacaatgag ttctactcgc agctctccaa tcccatgagc gcgctctgga actctttctc cacggctcag ctccagcaga tgcggaaccg gggctttcag tccgtgaacg atgtgtggca ggcggtgcgg gacagcccca tgttctttcc ccgtggccgg gcccgcacgt tgacgagaca gaaccccggc tttgatgcga ccacccgccg ggcggtctcc atcgggacca tccgcaacgc tcttgcgccg acggacttca tcacctttgg cagtggcaag accgccaacc acagtgagag cgccacccag ggcatcctgg aaagccagcc gcacaacaac gtgcacaaca acatcggcgg gttcatgcag gacctgctct ctcccaccga cccggtgttc ttcgcccacc attccaatat tgaccggctc tgggacgtgt ggacacgcaa acaacagcgt cttgggctgc ctacgctacc cacaggggcg aacctgccgc tgtgggccaa tgagcctttc ctcttcttca tcgggccgga cggcaaaccg gtggcgaaga acaaggcggg tgactacgcc acgatcggag actttgacta taactacgag cctggctccg gtgaggcggt gattccagct gccagccgcc ctggcgaaat gaacaacaag gtgtggctgg gcacgctcgg ggctgcagtg cccaacttta gtgcctccgc ccgcgctgat gtgatggtgc ctgaagccgt gccggaggcc gccatgaagg cggatggtcc cgcagttttc gccaagatca ccattgcgcc gcccatggat gtggccgggg tggagtttca cgtgctggtg aatccgccgg agaatgtgag ccatgtggac tttgattccc caagtttcgc gggaacgttc agcgtctttg gcaaacagct cggcgggcat aaaaatcagc ccctgagctt cctcatgccg ctgaccgagg cggtgaaaaa gctgcaggag accaacgaac tcaagcctgg ccagccgctg cgggtgcagg tggtggcaga aaggaaagga gtaaatttga cgcctttgca ggccaaggtg tcagagatct cagtgggcac cttctaa

SEQ ID NO. 9: DNA sequence of recombinant mature domain

gcaaa ataccaccgg ctcaatttac aaaaccctgc ggctgcacct ttcctggaga gctacaagaa ggccatcacc gtcatgctgc agctcccgcc ctcggatgcg cgcaactggt accggaacgc cttcatccac acgctggact gcccgcatgg gaactggtgg tttgtcgtgt ggcaccgtgg ctacaccggc tggtttgagc gcacggtccg cgagctcagt ggggacccca acttcgcctt tccttattgg gactggacgg cgctgccgca ggttccggac tctttcttca atggtgtgct ggaccccaac aatcctgcct tcattgccag ctacaatgag ttctactcgc agctctccaa tcccatgagc gcgctctgga actctttctc cacggctcag ctccagcaga tgcggaaccg gggctttcag tccgtgaacg atgtgtggca ggcggtgcgg gacagcccca tgttctttcc ccgtggccgg gcccgcacgt tgacgagaca gaaccccggc tttgatgcga ccacccgccg ggcggtctcc atcgggacca tccgcaacgc tcttgcgccg acggacttca tcacctttgg cagtggcaag accgccaacc acagtgagag cgccacccag ggcatcctgg aaagccagcc gcacaacaac gtgcacaaca acatcggcgg gttcatgcag gacctgctct ctcccaccga cccggtgttc ttcgcccacc attccaatat tgaccggctc tgggacgtgt ggacacgcaa acaacagcgt cttgggctgc ctacgctacc cacaggggcg aacctgccgc tgtgggccaa tgagcctttc ctcttcttca tcgggccgga cggcaaaccg gtggcgaaga acaaggcggg tgactacgcc acgatcggag actttgacta taactacgag cctggctccg gtgaggcggt gtaa

While there are shown and described presently preferred embodiments of the invention, it is to be distinctly understood that the invention is not limited thereto but may be otherwise variously embodied and practiced within the scope of the following claims. The fol-lowing examples further illustrate the invention.

Example 1

Cloning and expression of V. spinosum tyrosinase

a) Pro-tyrosinase cloning, expression and purification.

V. spinosum (Strain No. 4136) was obtained from DSMZ GmbH and cultured under the conditions recommended by the sup-plier. See table 1 for the sequence of the various primers used in the making of the tyrosinase constructs. The primers VerrucFPOl and VerrucRPOl were designed using the draft genome of V. spinosum, available from TIGR (Project ID: 10620); Pubmed genome ID NZ_ABIZ00000000. Primers VerrucFPOl and VerrucRPOl were then used to amplify out the DNA encoding the full length pre-pro-tyrosinase (Locus Tag VspiD_010100001190) directly from V. spinosum cells using PCR. The resulting PCR product was then digested with BamHI and ffindIII restriction enzymes and cloned into the pUC18 plasmid vector cut with the same two restriction enzymes. To construct a plasmid vector suitable for recombinant protein expression in E. coli, the primers VerrucRBSFP02 and VerrucRPOl were used to amplify out the pro-tyrosinase gene from the pUC18 plas-mid. The resulting PCR product was then digested with

EcoRI and HindIII restriction enzymes and sub-cloned into the pQE60 plasmid vector (Qiagen Inc.) cut with the same restriction enzymes.

The resulting pQE60-pro-tyrosinase plasmid was then transformed into the E. coli strain DH5α. For large scale protein expression and purification an overnight culture was grown from a single colony transformed with the plasmid in LB + 1% glucose + ampicillin lOOμg/mL. The over- night culture was used to inoculate (1:50) 50OmL of M9+ medium; M9 salts + 1% casein + 1% glycerol + lOOμM calcium + 2mM magnesium + lOOμM thiamine and lOOμg/mL am-picillin in a 2L Erlenmeyer flask. This culture was grown at 37°C with 180rpm shaking for 4-6h, OD600 * 0,5, then ImM IPTG and lOOμg/mL ampicillin was added and growth continued for another 18-2Oh, final OD600 * 5. At the end of this period cells were harvested by centrifugation and washed in 0,1M Tris-HCl pH 8, final wet cell weight = 10g/L. The washed cell pellet was re-suspended in the same Tris buffer using 2mL of buffer per gram wet weight of cells. To this was added lysozyme to lmg/mL and PMSF also to 500μM. Cells were then incubated for 1 hour on ice and frozen at -800C. Cells were then thawed and fur-ther PMSF was added to 500μM. The partially lysed cells were then sonicated with a Branson sonifier cell disrup-tor (Branson Ultrasonics corp.), equipped with a 13mm tip on 50% power using 5 20 second bursts. The sample was then centrifuged at 50,00Og for 30 minutes and the super-natant, the soluble fraction, either processed directly or stored at -800C. After thawing, an equal volume of 1OmM Tris-HCl buffer plus 3M ammonium sulphate pH8 was added to the soluble fraction. The sample was then loaded onto a phenyl sepharose fast flow column and protein was eluted with a step gradient from 1,5M, 1,2M, 0,9M, 0,6M, 0,3M to OM ammonium sulphate, running buffer 1OmM Tris-HCl pH8. The peak tyrosinase containing fractions (0.6M and 0.3M step) were pooled and were desalted by passing over a G25 column, running buffer 1OmM Tris-HCl pH8. The sample was then passed over a Q-sepharose column and the unbound fraction containing tyrosinase collected, running buffer 1OmM Tris-HCl pH8. Tyrosinase containing fractions were then pooled concentrated to 20mg/mL using a

Vivaspin20 centrifugal concentrator (Sartorius Stedium Biotech S.A.) and loaded onto Superdex 75 16/60 gel filtration column (GE healthcare) , 12OmL bed volume, running buffer 1OmM Tris-HCl pH8 plus 0.1M NaCl. Tyrosinase con-taining fractions were then pooled, concentrated to

20mg/mL and stored at -800C in lOOμL aliquots. All purification steps were performed using an AKTA purifier 100 FPLC (GE healthcare) . The final yield of purified protein, depending on the form or mutant being expressed, ranged from 50 to 100 mg per litre of cells.

b) Digestion of pro-tyrosinase to mature form Trypsin digest of purified pro-tyrosinase was performed by dissolving 20μg of proteomics grade porcine trypsin (Sigma-Aldrich) in 50μL of ImM HCl and mixing it with an aliquot of pro-tyrosinase («10mg) , final volume ImL in 0. IM Tris-HCl pH 8.0. The sample was then incubated at room temperature for up to 24 hours, after which time the protein was purified using the aforementioned Superdex 75 16/60 gel filtration column and/or analyzed using an SDS polyacrylamide gel (fig. 5) . The increase in activity due to trypsin digest is shown in fig. 6.

c) Recombinant mature form

To construct a plasmid vector suitable for recombinant protein expression in E. coli, the primers

VerrucRBSFP02 and VerrucRP02 were used to amplify out the mature tyrosinase gene from the pUC18-protyrosinase plasmid. The resulting PCR product was then digested with EcoRl and HindIII restriction enzymes and sub-cloned into the pQE60 vector (Qiagen inc.) cut with the same restriction enzymes. The protein was then expressed and purified in an identical manner to the pro-tyrosinase.

Exam.ple 2

Characterization of V. spinosum recombinant tyrosinase

Enzyme activity as a function of pH (0.1M sodium phos-phate pH 5 to 8,5) was monitored by dopachrome formation at 475 nm using a molar extinction co-efficient of 3600 M-1Cm"1. Each reaction contained, lOμL of 0,6mg/mL L-DOPA, lOμL of 50mg/mL L-tyrosine, lOμL of 0,2mg/mL tyrosinase and 2970μL of 0,1M sodium phosphate buffer. Enzyme activ-ity as a function of temperature (10-800C) was recorded in an identical manner except the pH was fixed at 6,8. Kinetic characterization of L-tyrosine and L-DOPA oxidation was measured by following the formation of MBTH ad-ducts at 507nm, using a molar extinction co-efficient of 38,000 M-1Cm"1. Each reaction contained lOμL of 0,6mg/mL L-DOPA, lOμL of 50mg/mL L-tyrosine, lOμL of 0.2mg/mL tyrosinase and 1485μL of 0,2M sodium phosphate buffer pH 6,8 and 1485μL of 2,0mg/mL MBTH in 4% DMF.

All measurements were recorded at 300C, except in activ-ity as a function of temperature experiments, using a stirred Peltier assembly with the absorbance spectra being monitored on a Cary50 bio UV/vis spectrophotometer (Varian Inc.). The results are depicted in fig. 4.

Example 3

Examples of Cross-linking activity of recombinant V. spinosum tyrosinase

Protein cross-linking experiments were performed by incubating Iμl of lOmg/mL recombinant

V. spinosum tyrosinase with lOOμL of 2mg/mL substrate protein (see figure legend for details) in 0.1 M sodium phosphate buffer pH 7 plus either a further lOOμL of buffer or lOOμL of 0.2mg/mL caffeic acid or of 0.2mg/mL phe-nol. Samples were then incubated for 1 hour at room temperature in a rotary mixer (18rpm) and an aliquot removed for analysis by SDS PAGE results shown in Fig.7.

Example 4

Use of V. spinosum recombinant tyrosinase to immobilise an industri-ally relevant enzyme, Candida antartica lipase B (CALB) .

CALB was immobilised into tyrosinase generated phenol aggregates by incubating 50μL of 0-lOmg/mL CALB plus 200μL of 0. IM sodium phosphate pH7.0 containing 0-50mg/mL phenol plus lμL of 0-lOmg/mL V. spinosum recombinant tyrosinase. Samples were then incubated at room temperature in a rotary mixer. After lhour 749uL of buffer were added a 2OuL sample of the mixture removed (for activity analysis) and the sample centrifuged at 20,00Og for 10 minutes. A 2OuL sample of the su-pernatant was removed and the pellet resuspended in 98OuL of buffer. A 2OuL sam-ple of the resuspended pellet was then taken for activity analysis. The pellet was washed a further 4 times and finally resuspended in 96OuL of buffer and a 2OuL sample taken for activity analysis.

Table 1. Primers used in this work. Restriction enzyme sites are underlined while start and stop codons are shown in bold.