WO2018002379A2

WO2018002379A2 - Enzymatic preparation of indigo dyes and in situ dyeing process

Info

Publication number: WO2018002379A2
Application number: PCT/EP2017/066410
Authority: WO
Inventors: Owik Matthias HEROLD-MAJUMDAR; Morten TOVBORG; Martin Hofrichter; Marzena Poraj-Kobielska; Henrick LUND
Original assignee: Novozymes A/S
Priority date: 2016-07-01
Filing date: 2017-07-03
Publication date: 2018-01-04
Also published as: WO2018002379A3

Abstract

The invention relates to use of a peroxygenase for preparing indigo dyes and an in situ method for dyeing a textile with indigo dyes.

Description

ENZYMATIC PREPARATION OF INDIGO DYES AND IN SITU DYEING PROCESS

Reference to a Sequence Listing

This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to use of a peroxygenase for preparing indigo dyes and an in situ method for dyeing a textile with indigo dyes.

Background

Indigo dye is an organic compound with a distinctive blue color. Historically, indigo was a natural dye extracted from plants, and this process was important economically because blue dyes were once rare. Nearly all indigo dye produced today - several thousand tons each year - is synthetic. Indigo is the blue of blue jeans.

The primary use for indigo is as a dye for cotton yarn, which is mainly for the production of denim cloth for blue jeans. On average, a pair of blue jean trousers requires 3 - 12 g of indigo. Small amounts are used for dyeing wool and silk.

Indigo is insoluble in water. Therefore, the dye powder must be reduced to the soluble leuco form using sodium dithionite as reduction agents and alkali (for example, sodium hydroxide). The textile is then dipped into the dyeing bath containing the reduced leuco indigo and subsequently exposed to air to let the indigo oxidize. Since the dye take up is limited, this process is repeated 6 - 8 times in consecutive arranged tanks. Here, the consumption of reduction agent is increasing over each step and waste water containing sulfate is produced.

Another possibility is the use of pre-reduced indigo which is reduced at the dye manufacturer and then shipped in inert containers to the dyeing house.

The chemistry used for producing indigo is quite harsh and not very environmentally friendly. It comprises the use of high temperature and molten alkali.

WO 2014/122109 discloses a method for preparing an indigo dye, comprising contacting an indole with a peroxygenase and a source of hydrogen peroxide.

However, there is still a need for a more efficient method for producing an indigo dye to improve the process economics. Furthermore, there is still a need for an improved method for dyeing a textile with an indigo dye. The present invention aims to meet the needs.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for dyeing a textile, comprising: contacting the textile with a peroxygenase, a source of hydrogen peroxide, and a substituted or unsubstituted indole, wherein the indole is unsubstituted or substituted once or twice in the benzene ring and wherein each substituent is independently selected from the group consisting of F, CI, Br, OH, NH₂, COOH, S0₃ ^", alkyl and alkoxy.

In another aspect, the present invention provides a method or process for converting a substituted or unsubstituted indole to the corresponding 2, 3-epoxy-1 /-/-indole or 3-hydroxy-1 /-/- indole, comprising contacting the indole with a peroxygenase having an increased selectivity for 3-hydroxy-1 /-/-indole, and a source of hydrogen peroxide; wherein the indole, 2,3-epoxy-1 /-/- indole or 3-hydroxy-1 /-/-indole may be substituted once or twice in the benzene ring (the aromatic ring) and wherein each substituent is independently selected from the group consisting of F, CI, Br, OH, NH₂, COOH, S0₃ ^", alkyl and alkoxy.

In another aspect, the invention provides a method for preparing a substituted or unsubstituted indigo dye, comprising contacting an indole with a peroxygenase having an increased selectivity for 3-hydroxy-1 /-/-indole, and a source of hydrogen peroxide; wherein the indole is unsubstituted or substituted once or twice in the benzene ring (the aromatic ring) and wherein each substituent is independently selected from the group consisting of F, CI, Br, OH, NH₂, COOH, S0₃ ^", alkyl and alkoxy.

In embodiments, the peroxygenase comprises or consists of an amino acid sequence which has at least 70% identity to the mature polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20. In other embodiments, the amino acid sequence comprises the motif: E-H-D-[G,A]-S-[L,I]-S-R.

Other aspects and embodiments of the invention are apparent from the description and examples. DEFINITIONS

Peroxygenase: The term "peroxygenase" means an enzyme exhibiting "unspecific peroxygenase" activity according to EC 1.1 1.2.1 , that catalyzes insertion of an oxygen atom from H2O2 into a variety of substrates, such as nitrobenzodioxole. For purposes of the present invention, peroxygenase activity is determined according to the procedure described in M. Poraj- Kobielska, M. Kinne, R. Ullrich, K. Scheibner, M. Hofrichter, "A spectrophotometric assay for the detection of fungal peroxygenases", Analytical Biochemistry (2012), vol. 421 , issue 1 , pp. 327- 329.

The peroxygenase of the present invention has at least 20%, preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95%, most preferably at least 100%, most preferably at least 120%, and even most preferably at least 150% of the peroxygenase activity of the mature polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20.

Mature polypeptide: The term "mature polypeptide" is defined herein as a polypeptide having peroxygenase activity that is in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In a preferred aspect, the mature polypeptide has the amino acid sequence shown in positions 1 to 328 of SEQ ID NO: 1 based on the N-terminal peptide sequencing data (Ullrich et al., 2004, Appl. Env. Microbiol. 70(8): 4575-4581 ), elucidating the start of the mature protein of AaeAPO peroxygenase enzyme.

Identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "identity".

For purposes of the present invention, the degree of identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics 16: 276-277; http://emboss.org), preferably version 5.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

Parent or parent peroxygenase: The term "parent" or "parent peroxygenase" means any polypeptide with peroxygenase activity to which an alteration is made to produce the enzyme variants of the present invention.

Variant: The term "variant" means a polypeptide having peroxygenase activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position The variants of the present invention have at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 120%, or at least 150% of the peroxygenase activity of the mature polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20.

Conventions for Designation of Variants

For purposes of the present invention, the mature polypeptide disclosed in SEQ ID NO: 19 is used to determine the corresponding amino acid residue in another peroxygenase. The amino acid sequence of another peroxygenase is aligned with the mature polypeptide disclosed in SEQ ID NO: 19, and based on the alignment, the amino acid position number corresponding to any amino acid residue in the mature polypeptide disclosed in SEQ ID NO: 19 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

Identification of the corresponding amino acid residue in another peroxygenase can be determined by an alignment of multiple polypeptide sequences using several computer programs including, but not limited to, MUSCLE (multiple sequence comparison by log - expectation; version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 51 1 -518; Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009, Methods in Molecular Biology 537: 39-64; Katoh and Toh, 2010, Bioinformatics 26: 1899-1900), and EMBOSS EMMA employing ClustalW (1.83 or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), using their respective default parameters.

When the other enzyme has diverged from the mature polypeptide of SEQ ID NO: 19 such that traditional sequence-based comparison fails to detect their relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615), other pairwise sequence comparison algorithms can be used. Greater sensitivity in sequence-based searching can be attained using search programs that utilize probabilistic representations of polypeptide families (profiles) to search databases. For example, the PSI-BLAST program generates profiles through an iterative database search process and is capable of detecting remote homologs (Atschul et al., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivity can be achieved if the family or superfamily for the polypeptide has one or more representatives in the protein structure databases. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffin and Jones, 2003, Bioinformatics 19: 874-881 ) utilize information from a variety of sources (PSI-BLAST, secondary structure prediction, structural alignment profiles, and solvation potentials) as input to a neural network that predicts the structural fold for a query sequence. Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919, can be used to align a sequence of unknown structure with the superfamily models present in the SCOP database. These alignments can in turn be used to generate homology models for the polypeptide, and such models can be assessed for accuracy using a variety of tools developed for that purpose.

For proteins of known structure, several tools and resources are available for retrieving and generating structural alignments. For example the SCOP superfamilies of proteins have been structurally aligned, and those alignments are accessible and downloadable. Two or more protein structures can be aligned using a variety of algorithms such as the distance alignment matrix (Holm and Sander, 1998, Proteins 33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998, Protein Engineering 1 1 : 739-747), and implementation of these algorithms can additionally be utilized to query structure databases with a structure of interest in order to discover possible structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present invention, the nomenclature described below is adapted for ease of reference. The accepted lUPAC single letter or three letter amino acid abbreviation is employed.

Substitutions. For an amino acid substitution, the following nomenclature is used: Original amino acid, position, substituted amino acid. Accordingly, the substitution of threonine at position 226 with alanine is designated as "Thr226Ala" or "T226A". Multiple mutations are separated by addition marks ("+"), e.g., "Gly205Arg + Ser41 1 Phe" or "G205R + S41 1 F", representing substitutions at positions 205 and 41 1 of glycine (G) with arginine (R) and serine (S) with phenylalanine (F), respectively.

Deletions. For an amino acid deletion, the following nomenclature is used: Original amino acid, position, *. Accordingly, the deletion of glycine at position 195 is designated as "Gly195*" or "G195*". Multiple deletions are separated by addition marks ("+"), e.g. , "Gly195* + Ser41 1 *" or "G195* + S41 1 *".

Insertions. For an amino acid insertion, the following nomenclature is used: Original amino acid, position, original amino acid, inserted amino acid. Accordingly the insertion of lysine after glycine at position 195 is designated "Gly195Glyl_ys" or "G195GK". An insertion of multiple amino acids is designated [Original amino acid, position, original amino acid, inserted amino acid #1 , inserted amino acid #2; etc.]. For example, the insertion of lysine and alanine after glycine at position 195 is indicated as "Gly195Glyl_ysAla" or "G195GKA".

In such cases the inserted amino acid residue(s) are numbered by the addition of lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue(s). In the above example, the sequence would thus be:

Multiple alterations. Variants comprising multiple alterations are separated by addition marks ("+"), e.g., "Arg170Tyr+Gly195Glu" or "R170Y+G195E" representing a substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively.

Different alterations. Where different alterations can be introduced at a position, the different alterations are separated by a comma, e.g., "Arg170Tyr,Glu" represents a substitution of arginine at position 170 with tyrosine or glutamic acid. Thus, "Tyr167Gly,Ala + Arg170Gly,Ala" designates the following variants:

"Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala", "Tyr167Ala+Arg170Gly", and "Tyr167Ala+Arg170Ala". Wild-type peroxygenase: The term "wild-type" peroxygenase means a peroxygenase expressed by a naturally occurring microorganism, such as a bacterium, yeast, or filamentous fungus found in nature.

Alkyl: The term "alkyl" in the present context represents a linear or branched hydrocarbon radical having 1 -3 carbon atoms. Representative examples include methyl, ethyl, n-propyl and /^'so-propyl.

Alkoxy: The term "alkoxy" in the present context represents a radical of the formula -OR, where R is alkyl as defined above. Representative examples include methoxy, ethoxy, n-propoxy and /^'so-propoxy.

Textile: The term "textiles" used herein is meant to include fibers, yarns, fabrics and garments.

Fabric can be constructed from fibers by weaving, knitting or non-woven operations. Weaving and knitting require yarn as the input whereas the non-woven fabric is the result of random bonding of fibers (paper can be thought of as non-woven). In the present context, the term "fabric" is also intended to include fibers and other types of processed fabrics.

According to the invention, the method of the invention may be applied to any textile known in the art (woven, knitted, or non-woven). In particular the process of the present invention may be applied to cellulose-containing or cellulosic textile, such as cotton, viscose, rayon, ramie, linen, lyocell (e.g., Tencel, produced by Courtaulds Fibers), or mixtures thereof, or mixtures of any of these fibers together with synthetic fibres (e.g., polyester, polyamid, nylon) or other natural fibers such as wool and silk., such as viscose/cotton blends, lyocell/cotton blends, viscose/wool blends, lyocell/wool blends, cotton/wool blends; flax (linen), ramie and other fabrics based on cellulose fibers, including all blends of cellulosic fibers with other fibers such as wool, polyamide, acrylic and polyester fibers, e.g., viscose/cotton/polyester blends, wool/cotton/polyester blends, flax/cotton blends etc.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides a method for dyeing a textile, comprising: contacting the textile with a peroxygenase, a source of hydrogen peroxide, and an indole, wherein the indole is unsubstituted or substituted once or twice in the benzene ring and wherein each substituent is independently selected from the group consisting of F, CI, Br, OH, NH₂, COOH, SO3^", alkyl and alkoxy.

In another aspect, the invention provides a method for preparing a substituted or unsubstituted indigo dye, comprising contacting an indole with a peroxygenase having an increased selectivity for 3-hydroxy-1 /-/-indole, and a source of hydrogen peroxide; wherein the indole is unsubstituted or substituted once or twice in the benzene ring (the aromatic ring) and wherein each substituent is independently selected from the group consisting of F, CI, Br, OH, NH2, COOH, SO3^", alkyl and alkoxy. Indole (or 1 /-/-indole) and indole derivates (such as indole, 6-bromoindole, 6-chloroindole, 4-chloroindole, 5-chloroindole and 5-bromoindole) can be epoxidized to 2, 3-epoxy-1 /-/-indole (and corresponding derivates) using peroxygenases ("APO/UPO") and hydrogen peroxide. 2, 3-Epoxy-1 /-/-indole further rearranges to 3-hydroxy-1 /-/- indole (may be referred to as indoxyl), which spontaneously is oxidized by oxygen (for example from air) to form indigo. The 1 /-/-indoles may contain 1 or 2, the same or different substituents R, where R may be F, CI, Br, OH, NH₂, COOH (carboxyl), S0₃ ^" (sulfonate), alkyl or alkoxy.

(1 ) 1 /-/-indoles; (2) 2, 3-epoxy-1 /-/-indoles; (3) 3,3-dihydro-1 /-/-indol-2-ones; (4) 3-hydroxy-1 /-/- indoles; (5) indigos: R = H (indigo), R = Br (6,6'-dibromoindigo), R = CI (6,6'-dichloroindigo). "APO" = aromatic peroxygenase, and "UPO" = unspecific peroxygenase.

Non-substituted 1 /-/-indole (or indole) produces indigo and 3,3-dihydro-1 /-/-indol-2-ones (may be referred to as 2-oxindol or 2-oxindole), while derivatives of indole produce a variety of purple, violet, and red colors. 6,6'-dibromoindigo is also known as tyrian purple.

Peroxygenase

The peroxygenase having an increased selectivity for 3-hydroxy-1 /-/-indole. In one embodiment, the peroxygenase has at least 1 10%, for example, at least 150%, at least 200%, at least 300%, at least 500%, at least 800%, or at least 1000% selectivity for 3-hydroxy-1 /-/-indole, compared with Agrocybe aegerita unspecific peroxygenase (/AaeUPO), when the peroxygenase is used to convert an indole to an indigo dye.

In another embodiment, the peroxygenase having an increased selectivity for 3-hydroxy- 1 /-/-indole has a decreased selectivity for 2-oxindole. In a specific embodiment, the peroxygenase having an increased selectivity for 3-hydroxy-1 /-/-indole has about 1 -90%, for example, about 5- 80%, about 10-70%, or about 15-60% selectivity for 2-oxindole, compared with Agrocybe aegerita unspecific peroxygenase (/AaeUPO), when the peroxygenase is used to convert an indole to an indigo dye.

The peroxygenase of the present invention is preferably recombinantly produced, and comprises or consists of an amino acid sequence having at least 70% identity, preferably at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to the mature polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20; preferably the mature polypeptide of SEQ ID NO:1 , 2 19 or 20.

In a preferred embodiment, the peroxygenase comprises an amino acid sequence represented by the motif: E-H-D-[G,A]-S-[L,I]-S-R (SEQ ID NO: 21 ).

In yet another embodiment, the peroxygenase comprises or consists of the mature polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20; or a fragment thereof having peroxygenase activity; preferably SEQ ID NO: 1 , SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 19 or the mature polypeptide of SEQ ID NO: 20.

Preferably, amino acid changes are of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, LeuA al, Ala/Glu, and Asp/Gly.

In addition to the 20 standard amino acids, non-standard amino acids (such as 4- hydroxyproline, 6-/V-methyl lysine, 2-aminoisobutyric acid, isovaline, and alpha-methyl serine) may be substituted for amino acid residues of a wild-type polypeptide. A limited number of non- conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for amino acid residues. "Unnatural amino acids" have been modified after protein synthesis, and/or have a chemical structure in their side chain(s) different from that of the standard amino acids. Unnatural amino acids can be chemically synthesized, and preferably, are commercially available, and include pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.

Essential amino acids in the parent polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (i.e., peroxygenase activity) to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699- 4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identities of essential amino acids can also be inferred from analysis of identities with polypeptides that are related to a polypeptide according to the invention.

Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991 , Biochem. 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 1: 127).

Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.

In an embodiment, the peroxygenase of the present invention may be a peroxygenase variant, which comprises an alteration at one or more positions corresponding to positions 5, 6, 1 1 , 19, 26, 28, 34, 39, 44, 52, 54, 55, 58, 59, 62, 65, 71 , 78, 83, 85, 86, 106, 107, 1 17, 120, 147, 151 , 152, 154, 156, 158, 162, 163, 165, 166, 167, 179, 183, 184, 195, 200, 201 , 206, 209, 215 and 216 of the mature polypeptide of SEQ ID NO: 19, wherein each alteration is independently a substitution, deletion or insertion and the variant has peroxygenase activity and wherein the variant has at least 70%, e.g. , at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably the mature polypeptide of SEQ ID NO: 1 , 2, 19 or 20.

In another embodiment, the variant of the present invention has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 1 , 2, 19 or 20.

In a further embodiment, the variant of the present invention is a variant of a parent peroxygenase, and wherein the parent peroxygenase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the mature polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably the mature polypeptide of SEQ ID NO: 1 , 2, 19 or 20.

In a further embodiment, the variant comprises one or more substitutions selected from the group consisting of

W5F

E6K,C

K1 1 N,C

M19L,G

H26N,Q,A

F28Y,L,V

R34K

K39Q

G44A

N52Q

T54A

A55L,V,F,I

A58L,V,F,S,I

L59I

F62V,L,A,G

M65T,L

N71 Q N78D

H83N

I85F,L,V

L86F

N106Q

K107C

T108A

W117F

S120G

F147T

M148L

L151 G,F,A,V

G152A.Q

N153D

I154V,T,A

F155I.V

T156C

G158A,T,F,L

E159D

A162F,L,I,V

Y163D.L

M165I.L

L166V

I167L

W179F

W183I.V.F

F184I.V.L

W195F

K200E

E201 S

V206H

A209T

Q215R, and

N216Q.

A55L N71 Q

1154V

W183I

W183V

E6K+K1 1 N+R34K+K39Q+G44A+A58S+M65T+N78D+S120G+F147T+N153D+M16 5I+L166V+1167L+K200E+E201 S+V206H+A209T+Q215R

G158A+W183V

G158A+W183I

L151A+G158A

L151A+W183V

K107C+T108A

I85V+M165L

M165L+W179F

W117F+W195F

E6C+W1 17F; and

L151A+W183I.

The total number of amino acid substitutions, deletions and/or insertions of the mature polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably the mature polypeptide of SEQ ID NO: 1 , 2, 19 or 20, is 1-20, e.g., 1 -10 and 1-5. In a preferable embodiment, the total number of amino acid substitutions, deletions and/or insertions of the mature polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20; preferably the mature polypeptide of SEQ ID NO:1 , 2, 19 or 20; is at most 10, preferably at most 9, more preferably at most 8, more preferably at most 7, more preferably at most 6, more preferably at most 5, more preferably at most 4, even more preferably at most 3, most preferably at most 2, and even most preferably at most 1 .

The concentration of peroxygenase is typically 0.001 mg/ml to 50 mg/ml, preferably 0.002 mg/ml to 10 mg/ml, more preferably 0.005 mg/ml to 10 mg/ml, and most preferably 0.008 mg/ml to 5 mg/ml.

Preparation of Variants

The present invention also relates to methods for obtaining a variant having peroxygenase activity, comprising: (a) introducing into a parent peroxygenase an alteration at one or more (e.g., several) positions corresponding to positions 5, 6, 1 1 , 19, 26, 28, 34, 39, 44, 52, 54, 55, 58, 59, 62, 65, 71 , 78, 83, 85, 86, 106, 107, 1 17, 120, 147, 151 , 152, 154, 156, 158, 162, 163, 165, 166, 167, 179, 183, 184, 195, 200, 201 , 206, 209, 215 and 216 of the mature polypeptide of SEQ ID NO: 19, wherein the variant has peroxygenase activity; and (b) recovering the variant.

The variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g., several) mutations are introduced at one or more defined sites in a polynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis involving the cleavage by a restriction enzyme at a site in the plasmid comprising a polynucleotide encoding the parent and subsequent ligation of an oligonucleotide containing the mutation in the polynucleotide. Usually the restriction enzyme that digests the plasmid and the oligonucleotide is the same, permitting sticky ends of the plasmid and the insert to ligate to one another. See, e.g., Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methods known in the art. See, e.g., U.S. Patent Application Publication No. 2004/0171 154; Storici et al., 2001 , Nature Biotechnol. 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the present invention. There are many commercial kits available that can be used to prepare variants.

Synthetic gene construction entails in vitro synthesis of a designed polynucleotide molecule to encode a polypeptide of interest. Gene synthesis can be performed utilizing a number of techniques, such as the multiplex microchip-based technology described by Tian et al. (2004, Nature 432: 1050-1054) and similar technologies wherein oligonucleotides are synthesized and assembled upon photo-programmable microfluidic chips.

Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991 , Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204) and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner ei a/., 1988, DMA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

Semi-synthetic gene construction is accomplished by combining aspects of synthetic gene construction, and/or site-directed mutagenesis, and/or random mutagenesis, and/or shuffling. Semi-synthetic construction is typified by a process utilizing polynucleotide fragments that are synthesized, in combination with PCR techniques. Defined regions of genes may thus be synthesized de novo, while other regions may be amplified using site-specific mutagenic primers, while yet other regions may be subjected to error-prone PCR or non-error prone PCR amplification. Polynucleotide subsequences may then be shuffled.

Source of hydrogen peroxide

The hydrogen peroxide required by the peroxygenase may be provided as an aqueous solution of hydrogen peroxide or a hydrogen peroxide precursor for in situ production of hydrogen peroxide. Any solid entity which liberates upon dissolution a peroxide, which is useable by peroxygenase, can serve as a source of hydrogen peroxide. Compounds which yield hydrogen peroxide upon dissolution in water or an appropriate aqueous based medium include but are not limited to metal peroxides, percarbonates, persulphates, perphosphates, peroxyacids, alkyperoxides, acylperoxides, peroxyesters, urea peroxide, perborates and peroxycarboxylic acids or salts thereof.

Another source of hydrogen peroxide is a hydrogen peroxide generating enzyme system, such as an oxidase together with a substrate for the oxidase. Examples of combinations of oxidase and substrate comprise, but are not limited to, amino acid oxidase (see e.g. US 6,248,575) and a suitable amino acid, glucose oxidase (see e.g. WO 95/29996) and glucose, lactate oxidase and lactate, galactose oxidase (see e.g. WO 00/50606) and galactose, and aldose oxidase (see e.g. WO 99/31990) and a suitable aldose. Another hydrogen peroxide generating enzyme system is disclosed in WO 2008/051491.

By studying EC 1.1.3._, EC 1.2.3._, EC 1.4.3._, and EC 1.5.3._ or similar classes (under the International Union of Biochemistry), other examples of such combinations of oxidases and substrates are easily recognized by one skilled in the art.

Alternative oxidants which may be used with peroxygenases is oxygen combined with a suitable hydrogen donor like ascorbic acid, dehydroascorbic acid, dihydroxyfumaric acid or cysteine. An example of such oxygen hydrogen donor system is described by Pasta et al., Biotechnology & Bioengineering, (1999) vol. 62, issue 4, pp. 489-493.

Hydrogen peroxide or a source of hydrogen peroxide may be added at the beginning of or during the method of the invention (during the reaction), e.g., as one or more separate additions or dosages of hydrogen peroxide; or continuously as fed-batch addition. If hydrogen peroxide is added during the reaction, for example as 1 mmole/min or more, the amount of hydrogen peroxide used in the reaction may correspond to a total concentration of several moles/l, depending on how long the reaction is continued. Such considerations are well known in the art, and well within the skills of a skilled person. Typical amounts of hydrogen peroxide (concentrations of hydrogen peroxide, which may be supplemented with more hydrogen peroxide when depleted during the reaction) correspond to levels of from 0.001 mM to 25 mM, preferably to levels of from 0.005 mM to 5 mM, and particularly to levels of from 0.01 to 1 mM or 0.02 to 2 mM hydrogen peroxide. Hydrogen peroxide may also be used in an amount corresponding to levels of from 0.1 mM to 25 mM, preferably to levels of from 0.5 mM to 15 mM, more preferably to levels of from 1 mM to 10 mM, and most preferably to levels of from 1 mM to 8 mM hydrogen peroxide.

Methods, Compositions and Uses

In a conventional dyeing process with indigo dyes, high temperature, sodium hydroxide

(high concentration) and sodium dithionite are used to produce an indigo dye, and indigo dye is further used to dye the textile. This is not environmentally friendly, and the process is time and cost consuming. By the method of the present invention, the textile is dyed during production of dye in situ with a peroxygenase. So the method of the present invention is environmentally friendly, easy to handle, and time and cost efficient. The method of the present invention is completely transformative and will change the conventional dyeing process.

In an embodiment, the in situ dyeing method of the present invention comprises:

(a) contacting the textile with a peroxygenase, and an indole; and

(b) adding a source of hydrogen peroxide in one or more dosages.

In another embodiment, the in situ dyeing method of the present invention comprises:

(a) contacting the textile with a source of hydrogen peroxide, and an indole; and

(b) adding a peroxygenase.

In another aspect, the present invention provides a method for converting a substituted or unsubstituted indole to the corresponding 2, 3-epoxy-1 /-/-indole or 3-hydroxy-1 /-/-indole (or 3,3- dihydro-1 /-/-indol-2-one), comprising contacting the indole with a peroxygenase having an increased selectivity for 3-hydroxy-1 /-/-indole, and a source of hydrogen peroxide; wherein the indole, 2, 3-epoxy-1 /-/-indole, or 3-hydroxy-1 /-/-indole (or 3,3-dihydro-1 /-/-indol-2-one) may be substituted once or twice in the benzene ring and wherein each substituent is independently selected from the group consisting of F, CI, Br, OH, NH2, COOH, SO3^", alkyl and alkoxy.

In another aspect, the invention provides a method for preparing a substituted or unsubstituted indigo dye, comprising contacting an indole with a peroxygenase having an increased selectivity for 3-hydroxy-1 /-/-indole, and a source of hydrogen peroxide; wherein the indole is unsubstituted or substituted once or twice in the benzene ring and wherein each substituent is independently selected from the group consisting of F, CI, Br, OH, NH2, COOH, SO3^", alkyl and alkoxy. In an embodiment, the method is carried out in the presence of oxygen.

The present invention provides an environmentally friendly alternative for producing indigo dyes by using enzymes with less by-product. It does not require any harsh reaction conditions, like high temperatures or highly acid/alkaline pH. It is industrially applicable considering the yield of the indigo dyes.

In an embodiment, the peroxygenase comprises or consists of an amino acid sequence which has at least 70% identity to the mature polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20. In preferred embodiments, the peroxygenase comprises or consists of an amino acid sequence having at least 70% identity to the mature polypeptide of SEQ ID NO: 1 , 2, 19 or 20. In more preferred embodiments, the peroxygenase comprises or consists of the mature polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20; or a fragment thereof having peroxygenase activity. In other embodiments, the amino acid sequence of the peroxygenase comprises the motif: E-H-D-[G,A]- S-[L,I]-S-R (SEQ ID NO: 21 ).

In an embodiment, the peroxygenase may be a peroxygenase variant, which comprises an alteration at one or more positions corresponding to positions 5, 6, 1 1 , 19, 26, 28, 34, 39, 44, 52, 54, 55, 58, 59, 62, 65, 71 , 78, 83, 85, 86, 106, 107, 1 17, 120, 147, 151 , 152, 154, 156, 158, 162, 163, 165, 166, 167, 179, 183, 184, 195, 200, 201 , 206, 209, 215 and 216 of the mature polypeptide of SEQ ID NO: 19, wherein each alteration is independently a substitution, deletion or insertion and the variant has peroxygenase activity and wherein the variant has at least 70% but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably the mature polypeptide of SEQ ID NO: 1 , 2, 19 or 20.

In an embodiment, the process of the present invention is flushed with nitrogen; preferably a source of hydrogen peroxide and the substituted or unsubstituted indole are mixed and flushed with nitrogen.

The nitrogen used in the present invention may be nitrogen from the air (from the atmosphere) or a nitrogen precursor for in situ production of nitrogen. Nitrogen may be added, e.g. as pressurized atmospheric air or as pure pressurized N2. Alternatively, nitrogen precursors may be inherently present and/or added to the effluent and which, upon dissociation or reduction, provide an in situ source of nitrogen.

In a further embodiment, the method of the present invention is carried out under normal atmospheric conditions.

In a further embodiment, the method of the present invention is carried out at pH 7-13, preferably pH 8-12, more preferably pH 9-1 1. In an even further embodiment, the method of the present invention is carried out in Britton- Robinson buffer, TRIS buffer or water.

The oxygen used to convert 3-hydroxy-1 /-/-indoles to indigos may be oxygen from the air (from the atmosphere) or an oxygen precursor for in situ production of oxygen. In many industrial applications, oxygen from the air will usually be present in sufficient quantity. If more O2 is needed, additional oxygen may be added, e.g. as pressurized atmospheric air or as pure pressurized O2. Alternatively, oxygen precursors such as peroxides may be inherently present and/or added to the effluent and which, upon dissociation or reduction, provide an in situ source of oxygen.

The invention also provides for use of a peroxygenase, a source of hydrogen peroxide, and an indole for in situ dyeing of a textile, wherein the indole is unsubstituted or substituted once or twice in the benzene ring and wherein each substituent is independently selected from the group consisting of F, CI, Br, OH, NH₂, COOH, S0₃ ^", alkyl and alkoxy.

The invention further provides for use of a peroxygenase having an increased selectivity for 3-hydroxy-1 /-/-indole for preparing an indigo dye, 2, 3-epoxy-1 /-/-indole or 3-hydroxy-1 /-/-indole, which is unsubstituted or substituted once or twice in the benzene ring(s), and wherein each substituent is independently selected from the group consisting of F, CI, Br, OH, NH₂, COOH, SO3^", alkyl and alkoxy.

In an embodiment, each substituent is independently selected from the group consisting of CI, Br, OH, NH2, COOH, and SO3^". Preferably, each substituent is independently selected from the group consisting of CI, Br, OH, NH2, and SO3^"; more preferably CI and Br.

The methods according to the invention may be carried out at a temperature between 10 and 90 degrees Celsius, preferably between 15 and 80 degrees Celsius, more preferably between 20 and 80 degrees Celsius, even more preferably between 20 and 70 degrees Celsius, even more preferably between 20 and 60 degrees Celsius, most preferably between 30 and 60 degrees Celsius, and in particular between 40 and 60 degrees Celsius.

The in situ methods of dyeing a textile of the invention may employ a treatment time of from 10 minutes to 240 minutes, preferably from 20 minutes to 180 minutes, more preferably from 30 minutes to 150 minutes, more preferably from 50 minutes to 140 minutes and in particular from 60 minutes to 120 minutes.

The method for converting a substituted or unsubstituted indole to the corresponding 2,3- epoxy-1 /-/-indole or 3-hydroxy-1 /-/-indole (or 3,3-dihydro-1 /-/-indol-2-one) or the method for preparing a substituted or unsubstituted indigo dye of the invention may employ a treatment time of from 5 minutes to 120 minutes, preferably from 5 minutes to 90 minutes, more preferably from 5 minutes to 60 minutes, more preferably from 5 minutes to 45 minutes, and in particular from 5 minutes to 30 minutes.

The invention is further defined in the following paragraphs:

[1 ]. A method for dyeing a textile, comprising: contacting the textile with a peroxygenase, a source of hydrogen peroxide, and an indole, wherein the indole is unsubstituted or substituted once or twice in the benzene ring and wherein each substituent is independently selected from the group consisting of F, CI, Br, OH, NH₂, COOH, S0₃ ^", alkyl and alkoxy.

[2]. The method of paragraph 1 , wherein the peroxygenase has an increased selectivity for 3-hydroxy-1 /-/-indole; preferably the peroxygenase has at least 1 10%, for example, at least 150%, at least 200%, at least 300%, at least 500%, at least 800%, or at least 1000% selectivity for 3- hydroxy-1 /-/-indole, compared with Agrocybe aegerita unspecific peroxygenase, when the peroxygenase is used to convert the indole to an indigo dye.

[3]. The method of paragraph 1 or 2, wherein each substituent is independently selected from the group consisting of CI, Br, OH, NH₂, COOH, and S0₃ ^".

[4]. The method of paragraph 3, wherein each substituent is independently selected from the group consisting of CI, Br, OH, NH2, and SO3^".

[5]. The method paragraph 1 , wherein the indole is an indole or an indole substituted with

Br.

[6]. The method of any of paragraphs 1 -5, comprising:

(a) contacting the textile with a peroxygenase, and an indole; and

(b) adding a source of hydrogen peroxide in one or more dosages.

[7]. The method of any of paragraphs 1 -5, comprising:

(b) adding a peroxygenase.

[8]. The method of any of paragraphs 1-7, wherein the peroxygenase comprises or consists of an amino acid sequence which has at least 70% identity, e.g., at least 75% identity, or at least 80% identity, preferably at least 85% identity, more preferably at least 90% identity, further more preferably at least 95% identity, most preferably at least 97% identity, and in particular at least 99% identity to the mature polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14,

15, 16, 17, or 18, 19 or 20.

[9]. The method of any of paragraphs 1-8, wherein the peroxygenase comprises or consists of an amino acid sequence having at least 70% identity, e.g., at least 75% identity, or at least 80% identity, preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95% identity, most preferably at least 97% identity, and in particular at least 99% identity to the amino acid sequence of SEQ ID NO: 1 , SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 19 or the mature polypeptide of SEQ ID NO: 20.

[10]. The method of any of paragraphs 1-9, wherein the amino acid sequence of the peroxygenase comprises the motif: E-H-D-[G,A]-S-[L,I]-S-R (SEQ ID NO: 21 ).

[1 1]. The method of any of paragraphs 1-10, wherein the peroxygenase comprises or consists of the mature polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15,

16, 17, 18, 19 or 20; or a fragment thereof having peroxygenase activity. [12]. The method of any of paragraphs 1-1 1 , wherein the peroxygenase is a peroxygenase variant, comprising an alteration at one or more positions corresponding to positions 5, 6, 1 1 , 19, 26, 28, 34, 39, 44, 52, 54, 55, 58, 59, 62, 65, 71 , 78, 83, 85, 86, 106, 107, 1 17, 120, 147, 151 , 152, 154, 156, 158, 162, 163, 165, 166, 167, 179, 183, 184, 195, 200, 201 , 206, 209, 215 and 216 of the mature polypeptide of SEQ ID NO: 19, wherein each alteration is independently a substitution, deletion or insertion and the variant has peroxygenase activity and wherein the variant has at least 70%, e.g. , at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably the mature polypeptide of SEQ ID NO: 1 , 2, 19 or 20.

[13]. The method of 12, wherein the variant has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 1 , 2, 19 or 20.

[14]. The method of paragraph 12 or 13, wherein the variant is a variant of a parent peroxygenase, and wherein the parent peroxygenase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the mature polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably the mature polypeptide of SEQ ID NO: 1 , 2, 19 or 20.

[15]. The method of any of paragraphs 12-14, wherein the variant comprises one or more substitutions selected from the group consisting of

W5F

E6K,C

K1 1 N,C

M19L,G

H26N,Q,A

F28Y,L,V

R34K

K39Q

G44A

N52Q

T54A

A55L,V,F,I

A58L,V,F,S,I

L59I

F62V,L,A,G M65T.L

N71 Q

N78D

H83N

I85F,L,V

L86F

N106Q

K107C

T108A

W117F

S120G

F147T

M148L

L151 G,F,A,V

G152A.Q

N153D

I154V,T,A

F155I.V

T156C

G158A,T,F,L

E159D

A162F,L,I,V

Y163D.L

M165I.L

L166V

I167L

W179F

W183I.V.F

F184I.V.L

W195F

K200E

E201 S

V206H

A209T

Q215R, and [16]. The method of any of paragraphs 12-15, wherein the variant comprises one or more substitutions selected from the group consisting of

A55L

N71 Q

1154V

W183I

W183V

E6K+K1 1 N+R34K+K39Q+G44A+A58S+M65T+N78D+S120G+F147T+N153D+M165I+ L166V+1167L+K200E+E201 S+V206H+A209T+Q215R

G158A+W183V

G158A+W183I

L151A+G158A

L151A+W183V

K107C+T108A

I85V+M165L

M165L+W179F

W117F+W195F

E6C+W1 17F; and

L151A+W183I.

[17]. The method of any paragraphs 1-16, which is flushed with nitrogen; preferably a source of hydrogen peroxide and the substituted or unsubstituted indole are mixed and flushed with nitrogen.

[18]. The method of any of paragraphs 1 -17, which is carried out under normal atmospheric conditions.

[19]. The method of any of paragraphs 1-18, which is carried out at pH 7-13, preferably pH 8-12, more preferably pH 9-1 1.

[20]. The method of any of paragraphs 1 -19, which is carried out in Britton-Robinson buffer, TRIS buffer or water.

[21]. A method for converting a substituted or unsubstituted indole to the corresponding 2,3- epoxy-1 /-/-indole or 3-hydroxy-1 /-/-indole, comprising contacting the indole with a peroxygenase having an increased selectivity for 3-hydroxy-1 /-/-indole, and a source of hydrogen peroxide; wherein the indole, 2, 3-epoxy-1 /-/-indole, or 3-hydroxy-1 /-/-indole is substituted once or twice in the benzene ring and wherein each substituent is independently selected from the group consisting of F, CI, Br, OH, NH₂, COOH, S0₃ ^", alkyl and alkoxy.

[22]. A method for preparing a substituted or unsubstituted indigo dye, comprising contacting an indole with a peroxygenase having an increased selectivity for 3-hydroxy-1 /-/-indole, and a source of hydrogen peroxide; wherein the indole is unsubstituted or substituted once or twice in the benzene ring and wherein each substituent is independently selected from the group consisting of F, CI, Br, OH, NH₂, COOH, S0₃ ^", alkyl and alkoxy.

[23]. The method of any of paragraphs 21-22, wherein each substituent is independently selected from the group consisting of CI, Br, OH, NH2, COOH, and SO3^".

[24]. The method of paragraph 23, wherein each substituent is independently selected from the group consisting of CI, Br, OH, NH2, and SO3^".

[25]. The method of any of paragraphs 21 -24, wherein the substituted or unsubstituted indole is the indole or indole substituted with Br.

[26]. The method of any of paragraph 21-25, comprising:

(a) contacting the textile with a peroxygenase, and a substituted or unsubstituted indole; and

(b) adding a source of hydrogen peroxide in one or more dosages.

[27]. The method of any of paragraphs 21 -25, comprising:

(a) contacting the textile with a source of hydrogen peroxide, and a substituted or unsubstituted indole; and

(b) adding a peroxygenase.

[28]. The method of any of paragraphs 21 -27, wherein the peroxygenase comprises or consists of an amino acid sequence which has at least 70% identity, e.g., at least 75% identity, or at least 80% identity, preferably at least 85% identity, more preferably at least 90% identity, further more preferably at least 95% identity, most preferably at least 97% identity, and in particular at least 99% identity to the mature polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, or 18, 19 or 20.

[29]. The method of any of paragraphs 21 -28, wherein the peroxygenase comprises or consists of an amino acid sequence having at least 70% identity, e.g., at least 75% identity, or at least 80% identity, preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95% identity, most preferably at least 97% identity, and in particular at least 99% identity to the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 19 or the mature polypeptide of SEQ ID NO: 20.

[30]. The method of any of paragraphs 21-29, wherein the amino acid sequence of the peroxygenase comprises the motif: E-H-D-[G,A]-S-[L,I]-S-R (SEQ ID NO: 21 ).

[31]. The method of any of paragraphs 21 -30, wherein the peroxygenase comprises or consists of the mature polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20; or a fragment thereof having peroxygenase activity.

[32]. The method of any of paragraphs 21-31 , wherein the peroxygenase is a peroxygenase variant, comprising an alteration at one or more positions corresponding to positions 5, 6, 1 1 , 19, 26, 28, 34, 39, 44, 52, 54, 55, 58, 59, 62, 65, 71 , 78, 83, 85, 86, 106, 107, 1 17, 120, 147, 151 , 152, 154, 156, 158, 162, 163, 165, 166, 167, 179, 183, 184, 195, 200, 201 , 206, 209, 215 and 216 of the mature polypeptide of SEQ ID NO: 19, wherein each alteration is independently a substitution, deletion or insertion and the variant has peroxygenase activity and wherein the variant has at least 70%, e.g. , at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably the mature polypeptide of SEQ ID NO: 1 , 2, 19 or 20.

[33]. The method of paragraph 32, wherein the variant has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 1 , 2, 19 or 20.

[34]. The method of paragraph 32 or 33, wherein the variant is a variant of a parent peroxygenase, and wherein the parent peroxygenase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the mature polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably the mature polypeptide of SEQ ID NO: 1 , 2, 19 or 20.

[35]. The method of any of paragraphs 32-34, wherein the variant comprises one or more substitutions selected from the group consisting of

W5F

E6K,C

K1 1 N,C

M19L,G

H26N,Q,A

F28Y,L,V

R34K

K39Q

G44A

N52Q

T54A

A55L,V,F,I

A58L,V,F,S,I

L59I

F62V,L,A,G

M65T,L

N71 Q

N78D

H83N I85F,L,V

L86F

N106Q

K107C

T108A

W117F

S120G

F147T

M148L

L151 G,F,A,V

G152A.Q

N153D

I154V,T,A

F155I.V

T156C

G158A,T,F,L

E159D

A162F,L,I,V

Y163D.L

M165I.L

L166V

I167L

W179F

W183I.V.F

F184I.V.L

W195F

K200E

E201 S

V206H

A209T

Q215R, and

N216Q.

[36]. The method of any of paragraphs 32-35, wherein the variant comprises one or more substitutions selected from the group consisting of

A55L

N71 Q

1154V W183I

W183V

E6K+K1 1 N+R34K+K39Q+G44A+A58S+M65T+N78D+S120G+F147T+N153D+M165I+L1 66V+1167L+K200E+E201 S+V206H+A209T+Q215R

G158A+W183V

G158A+W183I

L151A+G158A

L151A+W183V

K107C+T108A

I85V+M165L

M165L+W179F

W117F+W195F

E6C+W1 17F; and

L151A+W183I.

[37]. The method of any paragraphs 21-36, which is flushed with nitrogen; preferably a source of hydrogen peroxide and the substituted or unsubstituted indole are mixed and flushed with nitrogen.

[38]. The method of any of paragraphs 21 -37, which is carried out under normal atmospheric conditions.

[39]. The method of any of paragraphs 21-38, which is carried out at pH 7-13, preferably pH

8-12, more preferably pH 9-1 1.

[40]. The method of any of paragraphs 21 -39, which is carried out in Britton-Robinson buffer, TRIS buffer, or water.

[41]. Use of a peroxygenase, a source of hydrogen peroxide, and an indole for in situ dyeing of a textile, wherein the indole is unsubstituted or substituted once or twice in the benzene ring and wherein each substituent is independently selected from the group consisting of F, CI, Br, OH, NH₂, COOH, S0₃-, alkyl and alkoxy.

[42]. Use of a peroxygenase having an increased selectivity for 3-hydroxy-1 /-/-indole for preparing an indigo dye, 2, 3-epoxy-1 /-/-indole or 3-hydroxy-1 /-/-indole, which is unsubstituted or substituted once or twice in the benzene ring(s), and wherein each substituent is independently selected from the group consisting of F, CI, Br, OH, NH2, COOH, SO3^", alkyl and alkoxy.

[43]. The use according to paragraph 41 or 42, wherein each substituent is independently selected from the group consisting of CI, Br, OH, NH2, and SO3^".

[44]. A peroxygenase variant, comprising an alteration at one or more positions corresponding to positions 5, 6, 1 1 , 19, 26, 28, 34, 39, 44, 52, 54, 55, 58, 59, 62, 65, 71 , 78, 83, 85, 86, 106, 107, 1 17, 120, 147, 151 , 152, 154, 156, 158, 162, 163, 165, 166, 167, 179, 183, 184, 195, 200, 201 , 206, 209, 215 and 216 of the mature polypeptide of SEQ ID NO: 19, wherein each alteration is independently a substitution, deletion or insertion and the variant has peroxygenase activity and wherein the variant has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably the mature polypeptide of SEQ ID NO: 1 , 2, 19 or 20.

[45]. The peroxygenase variant of paragraph 44, wherein the variant has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 1 , 2, 19 or 20.

[46]. The peroxygenase variant of paragraph 44 or 45, which is a variant of a parent peroxygenase, and wherein the parent peroxygenase has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the mature polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably the mature polypeptide of SEQ ID NO: 1 , 2, 19 or 20.

[47]. The peroxygenase variant of any of paragraphs 44-46, wherein the variant comprises one or more substitutions selected from the group consisting of

W5F

E6K,C

K1 1 N.C

M19L,G

H26N,Q,A

F28Y,L,V

R34K

K39Q

G44A

N52Q

T54A

A55L,V,F,I

A58L,V,F,S,I

L59I

F62V,L,A,G

M65T,L

N71 Q

N78D

H83N I85F,L,V

L86F

N 106Q

K107C

T108A

W1 17F

S120G

F147T

M148L

L151 G,F,A,V

G152A.Q

N 153D

I 154V,T,A

F155I.V

T156C

G158A,T,F,L

E159D

A162F,L,I,V

Y163D.L

M165I.L

L166V

I 167L

W179F

W183I.V.F

F184I.V.L

W195F

K200E

E201 S

V206H

A209T

Q215R, and

N216Q.

[48]. The peroxygenase variant of any of paragraphs 44-47, wherein the variant comprises one or more substitutions selected from the group consisting of

A55L

N71 Q

1154V W183I

W183V

G158A+W183V

G158A+W183I

L151A+G158A

L151A+W183V

K107C+T108A

I85V+M165L

M165L+W179F

W117F+W195F

E6C+W1 17F; and

L151A+W183I.

[49]. The peroxygenase variant of any of paragraphs 44-48, wherein the number of alterations is 1 -20, e.g., 1-10 and 1 -5, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations.

The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

EXAMPLES

LC-DAD-MS Method for the Analysis of Indole and Reaction Products

Equipment:

MicroDegasser G1379B

Binary pump G1312B

High Performance AutoSampler (HiP ALS) G1367E

Thermostatted Column Compartment (TCC) G1316C

Detector 1 : Diode Array detector (DAD) G4212B

Detector 2: Single Quadrupole Mass detector (MS) G6120B

Vacuum pump (VARIAN MS40+) 949-9241

Nitrogen generator (PEAK) NM32LA

Column Luna C18 250x2 mm 5 μιη particle

Method:

Time (min) MilliQ+0.1 % Formic acid Acetonitril+0.1 % Formic acid Flow (mL/min)

0 80 % 20 % 0.35

10 20 % 80 % 0.35

15 5 % 95 % 0.35 Column was heated to 40 °C.

10 μΙ_ Reaction Medium was injected after centrifuging (10 min, 10000 g). If needed dilutions were prepared. Analysis of compounds was done via standards and mass spectrum.

MS-Detector Settings:

Gas Temp: 350 °C

DryingGas: 12.0 L/min

Neb Pres: 35 psig

Scan: 40.00 - 280.00 m/z

Fragmentor: 70 V / 180 V

Chemicals used as buffers and substrates were commercial products of at least reagent grade.

Humicola insolens unspecific peroxygenase (HinUPO): disclosed as mature polypeptide of SEQ ID NO:2 in WO 2013/021061 , and as SEQ ID NO: 19 herein;

Daldinia caldariorum unspecific peroxygenase (DcaUPO) disclosed as mature polypeptide of SEQ ID NO:2 in WO 2014/056917, and as SEQ ID NO: 20 herein;

Agrocybe aegerita unspecific peroxygenase (AaeUPO) disclosed as mature polypeptide of SEQ ID NO: 1 in WO 2014/122109, and as SEQ ID NO: 1 herein;

Coprinopsis cinerea unspecific peroxygenase (Cc/^'UPO) disclosed as mature polypeptide of SEQ ID NO:2 in WO 2014/122109, and as SEQ ID NO: 2 herein.

Example 1 : Improved conversion and selectivity for an enzymatic conversion of indole to indigo using Humicola insolens unspecific peroxygenase (HinUPO) and Daldinia caldariorum unspecific peroxygenase (DcaUPO)

1 mM substrate (indole) was converted by 0.01 mg/mL peroxygenase in 1 ml. of 50 mM phosphate buffer and 1 mM Hydrogen peroxide.

A yellow color formation turning green and finally blue was observed shortly after reaction start indicating the formation of indigo dye. This is further supported by precipitation of blue dye and a turbid blue solution.

Table 1 : Results from LC-DAD-MS measurement. Substrate and major by-product concentrations after reaction are listed:

From the results, it can be seen that H/^'nUPO and DcaUPO increase the conversion of indole, and the selectivity towards the by-product 2-Oxindole is decreased using H/^'nUPO and DcaUPO compared to enzymes /AaeUPO and Cc/^'UPO. Example 2: Increased yield of indigo using nitrogen atmosphere

5.725 mM substrate (indole) were converted by 0.01 mg/mL Humicola insolens unspecific peroxygenase (HinUPO) and variants thereof in 10 mL of 10 mM Britton-Robinson (BR, composed of phosphoric acid, acetic acid, boric acid, titrated with NaOH) buffer at different atmospheric conditions ( ) and 2 mM Hydrogen peroxide for 2 hours at room temperature.

For the reaction in nitrogen atmosphere, prior to enzyme addition, the substrate, buffer, hydrogen peroxide mixture was flushed with nitrogen for 15 minutes. The reaction was started with the addition of enzyme and nitrogen was bubbled trough the reaction over the course of the reaction. The product was stirred open after reaction for 1 hour.

The reaction under normal atmospheric conditions was conducted in a closed vessel during the reaction and after reaction stirred open for 1 hour.

The reaction with aeration was conducted under constant air bubbling. The products were stirred open after reaction for 1 hour.

Quantification was performed using High Performance Liquid Chromatography (HPLC), diethylether extraction and sulfonation of the indigo. Thereby, formation of 2-Oxindole, unknown products and indigo was found.

From the results, it can be seen that H/^'nUPO and the variants thereof could convert indole to an indigo dye in high yield at nitrogen atmosphere, and the variant with A55L mutation worked better than the other tested enzymes. The best results in terms of obtained indigo production were received for variant with A55L mutation under normal atmospheric conditions.

Table 2: Production indigo for the wild type H/^'nUPO enzyme and variants thereof

Indigo Yield on Limiting Substrate

H/nUPO and Aeration

N₂ Normal atmospheric conditions

variants

Wild type 61 .9 % 24.4 % 10.0 %

A55L 49.0 % 65.7 % 42.3 %

N71 Q 43.7 % 17.5 % 16.0 %

1154V 33.7 % 17.9 % 1 1 .5 %

W183I 47.1 % 24.7 % 18.4 % W183V 43.6 % 23.1 % 19.6 %

Percentages are given on the limiting substrate. Percentages were calculated the following way: [lndigo]^*2/[H2O2]^*100 (Concentration of indigo times 2 divided by the concentration of hydrogen peroxide (limiting substrate in this case) times 100) Example 3: Conversion of indole to indigo by peroxygenases at alkaline pH

5.725 mM substrate (indole) were converted by 0.01 mg/mL H/^'nUPO in 10 mL of 5 mM

Britton-Robinson buffer at various pH (Table 3) and 2 mM Hydrogen peroxide. Prior to enzyme addition, the substrate, buffer, hydrogen peroxide mixture was flushed with nitrogen for 15 minutes. The reaction was started with the addition of enzyme.

Quantification was performed using HPLC and sulfonation of the indigo. Thereby, formation of 2-Oxindole, unknown products and indirubin was found.

From the results, it can be seen that the reaction has a broad pH optimum reaching far into the alkaline region, and thereby, giving robustness to the reaction and the opportunity to adjust the system.

Table 3: Enzymatic Indigo production at elevated pH

Example 4: Improved conversion at pH=10 in TRIS (Tris(hydroxymethyl)aminomethane) buffer

5.725 mM substrate (indole) were converted by 0.01 mg/mL H/^'nUPO in 10 mL of 5 mM TRIS buffer at various pH (Table 4) and 2 mM Hydrogen peroxide. Prior to enzyme addition, the substrate, buffer, hydrogen peroxide mixture was flushed with nitrogen for 15 minutes. The reaction was started with the addition of enzyme.

From the results, it can be seen that the reaction at pH 10 with TRIS buffer resulted in the highest conversion to indigo. This is unnatural for this enzyme and it is surprising to find the enzyme can work at such alkaline pH.

Table 4: Improved conversion of indole to indigo with TRIS buffer at pH 10

Example 5: Screening of Humicola insolens unspecific peroxygenase variants

5.0/1.0 mM indole (Table 5) were converted by 0.01 mg/mL H/^'nUPO and variants thereof indicated with different numbers in 96-well plate format with total reaction volume of 160 μΙ_. Reaction conditions were 5 mM TRIS pH=10 buffer, 2 mM H2O2, room temperature and shaking with 400 rpm on a plate shaker. The reaction was started with the addition of H2O2.

Reaction was stopped using 10 μΙ_ of a 1 :10 dilution of Terminox Supreme (Novozymes A/S) and shaking for 30 min. Subsequently, 1 10 μΙ_ 96 % H2SO4 were added and absorbance at 640 nm was measured immediately.

Quantification was performed by the use of an indigo standard curve.

From the results, it can be seen that the reaction of indole to indigo could be catalyzed by a wide range of variants of H/^'nUPO. Best picks are chosen to be validated via Liquid chromatography-mass spectrometry (LC-MS).

Table 5: Results from H/nUPO variants screening on indole at two different substrate concentrations

W183V 1 .0 0.187 ± 0.004

F28Y 5.0 0.073 ± 0.003

F28Y 1 .0 0.105 ± 0.005

F28L 5.0 0.073 ± 0.005

F28L 1 .0 0.108 ± 0.008

I85L 5.0 0.012 ± 0.001

I85L 1 .0 0.023 ± 0.000

I85V 5.0 0.040 ± 0.003

I85V 1 .0 0.076 ± 0.012

L151V 5.0 0.015 ± 0.001

L151V 1 .0 0.033 ± 0.003

W183F 5.0 0.037 ± 0.01 1

W183F 1 .0 0.005 ± 0.000

M19L 5.0 0.048 ± 0.010

M19L 1 .0 0.005 ± 0.000

E6K+K1 1 N+R34K+K39Q+G44A+A58S+M65T+N78D+ 5.0 0.003 ± 0.000 S120G+F147T+N153D+M165I+L166V+I167L+K200E+

E201 S+V206H+A209T+Q215R

E6K+K1 1 N+R34K+K39Q+G44A+A58S+M65T+N78D+ 1 .0 0.005 ± 0.001 S120G+F147T+N153D+M165I+L166V+I167L+K200E+

E201 S+V206H+A209T+Q215R

E6C 5.0 0.039 ± 0.027

E6C 1 .0 0.006 ± 0.001

I154A 5.0 0.048 ± 0.01 1

I154A 1 .0 0.017 ± 0.008

1154V 5.0 0.015 ± 0.001

1154V 1 .0 0.004 ± 0.001

F62V 5.0 0.007 ± 0.000

F62V 1 .0 0.004 ± 0.001

A55V 5.0 0.021 ± 0.007

A55V 1 .0 0.005 ± 0.000

G158A+W183V 5.0 0.005 ± 0.000

G158A+W183V 1 .0 0.003 ± 0.000

G158A+W183I 5.0 0.005 ± 0.001

G158A+W183I 1 .0 0.003 ± 0.000

L151A+G158A 5.0 0.003 ± 0.000

L151A+G158A 1 .0 0.003 ± 0.001

L151A+W183V 5.0 0.003 ± 0.000

L151A+W183V 1 .0 0.003 ± 0.000

H83N 5.0 0.021 ± 0.002 H83N 1.0 0.014 + 0.001

G152A 5.0 0.021 + 0.001

G152A 1.0 0.013 + 0.000

G152Q 5.0 0.022 + 0.005

G152Q 1.0 0.018 + 0.002

T156C 5.0 0.019 + 0.001

T156C 1.0 0.010 + 0.004

L86F 5.0 0.035 + 0.003

L86F 1.0 0.019 + 0.000

A55V 5.0 0.005 + 0.000

A55V 1.0 0.003 + 0.000

F62L 5.0 0.103 + 0.002

F62L 1.0 0.116 + 0.003

F62A 5.0 0.095 + 0.006

F62A 1.0 0.143 + 0.006

A58L 5.0 0.074 + 0.010

A58L 1.0 0.102 + 0.001

A58V 5.0 0.068 + 0.002

A58V 1.0 0.105 + 0.004

W117F 5.0 0.031 + 0.001

W117F 1.0 0.065 + 0.004

W195F 5.0 0.046 + 0.002

W195F 1.0 0.109 + 0.005

N106Q 5.0 0.059 + 0.001

N106Q 1.0 0.125 + 0.011

E159D 5.0 0.007 + 0.002

E159D 1.0 0.010 + 0.002

A162I 5.0 0.110 + 0.005

A162I 1.0 0.119 + 0.014

A162V 5.0 0.050 + 0.005

A162V 1.0 0.090 + 0.013

F184I 5.0 0.045 + 0.003

F184I 1.0 0.082 + 0.003

F184V 5.0 0.025 + 0.004

F184V 1.0 0.050 + 0.005

F28V 5.0 0.086 + 0.012

F28V 1.0 0.118 + 0.005

M65L 5.0 0.076 + 0.007

M65L 1.0 0.113 + 0.008 N52Q 5.0 0.057 + 0.003

N52Q 1.0 0.111 + 0.009

T73A 5.0 0.075 + 0.014

T73A 1.0 0.118 + 0.019

G158T 5.0 0.017 + 0.001

G158T 1.0 0.021 + 0.001

L151A 5.0 0.015 + 0.002

L151A 1.0 0.019 + 0.001

F184L 5.0 0.016 + 0.001

F184L 1.0 0.020 + 0.002

F62G 5.0 0.017 + 0.002

F62G 1.0 0.020 + 0.002

K11C 5.0 0.055 + 0.025

K11C 1.0 0.004 + 0.000

K107C+T108A 5.0 0.130 + 0.035

K107C+T108A 1.0 0.005 + 0.000

N216Q 5.0 0.024 + 0.010

N216Q 1.0 0.006 + 0.001

G158F 5.0 0.008 + 0.001

G158F 1.0 0.006 + 0.001

I85V+M165L 5.0 0.018 + 0.007

I85V+M165L 1.0 0.005 + 0.001

M165L+W179F 5.0 0.011 + 0.004

M165L+W179F 1.0 0.004 + 0.001

W117F+W195F 5.0 0.008 + 0.003

W117F+W195F 1.0 0.006 + 0.003

E6C+W117F 5.0 0.004 + 0.001

E6C+W117F 1.0 0.005 + 0.001

L151A+W183I 5.0 0.008 + 0.002

L151A+W183I 1.0 0.004 + 0.000

H26N 5.0 0.005 + 0.001

H26N 1.0 0.003 + 0.000

H26Q 5.0 0.018 + 0.004

H26Q 1.0 0.004 + 0.001

H26A 5.0 0.008 + 0.001

H26A 1.0 0.007 + 0.004

M19G 5.0 0.019 + 0.003

M19G 1.0 0.062 + 0.004

Y163D 5.0 0.006 + 0.000 Y163D 1 .0 0.048 + 0.003

Y163L 5.0 0.031 + 0.007

Y163L 1 .0 0.087 + 0.007

L59I 5.0 0.008 + 0.002

L59I 1 .0 0.020 + 0.002

A58I 5.0 0.007 + 0.000

A58I 1 .0 0.005 + 0.000

A55F 5.0 0.009 + 0.000

A55F 1 .0 0.010 + 0.001

A55I 5.0 0.193 + 0.004

A55I 1 .0 0.128 + 0.010

M148L 5.0 0.050 + 0.000

M148L 1 .0 0.070 + 0.003

M165L 5.0 0.195 + 0.007

M165L 1 .0 0.157 + 0.006

G158A 5.0 0.020 + 0.001

G158A 1 .0 0.064 + 0.003

G158L 5.0 0.051 + 0.003

G158L 1 .0 0.063 + 0.004

F155I 5.0 0.009 + 0.002

F155I 1 .0 0.01 1 + 0.000

F155V 5.0 0.006 + 0.001

F155V 1 .0 0.008 + 0.000

Standard error is given of 3 technical replica

Example 6: Upscaling of reaction to 100 ml_

5.725 mM indole were converted by 0.01 mg/mL H/^'nUPO in 100 mL of 5 mM TRIS buffer at pH 10 and 2 mM Hydrogen peroxide. Prior to enzyme addition, the substrate, buffer, hydrogen peroxide mixture was flushed with nitrogen for 15 minutes. The reaction was started with the addition of enzyme via a syringe.

Quantification was performed using HPLC and sulfonation of the indigo. Thereby, formation of 2-Oxindole and unknown products was found (Table 6).

Surprisingly, higher yields were obtained when increasing the scale of the reaction. It can also be seen that the reaction worked well at larger scale.

Table 6: Yields of upscale experiment. Percentages are given based on limiting substrate: Product Quantity

Indigo 72.4 %

2-Oxindole 21.8 %

Unknown products 5.80 %

Example 7: Improved conversion and selectivity for the enzymatic conversion of 6- bromoindole to Tyrian purple using Humicola insolens unspecific peroxygenase (H/nllPO)

Typical reaction mixtures (1.0 ml) contained purified H/^'nUPO (0.1 mg/mL and 0.3 mg/mL), substrate to be oxidized (6-bromoindole; 0.5-2.0 mM; stock solution 50/50 H2O/ACN = 5% end concentration), potassium phosphate buffer (50 mM, pH 7.0) and water. The reactions were started by the addition of limiting H2O2 (2.0-5.0 mM) and stirred at room temperature over half an hour. Rest product and 6-Br-2-oxindole were obtained, by use of HPLC, 6,6'-dibromoindigo by sulfonation. By the conversion with 0.1 mg/mL H/^'nUPO, about 60% of substrate was converted to 6,6'-dibromoindigo, 30% to 6-Br-2-oxindole. Rest was converted to other, unknown polymers. By the conversion with higher concentration of H/^'nUPO, the yield of 6,6'-dibromoindigo has no significant changed, and the 6-Br-2-oxindole was converted to other polymers.

Example 8: In situ dyeing process based on the conversion of 6-bromoindole to Tyrian purple catalyzed by Coprinopsis cinerea unspecific peroxygenase (Cc/UPO)

5 Liter of 100 mM potassium phosphate buffer (pH 7) was filled into a 25 liter steel tank. 2 g of substrate (6-bromoindole) was dissolved in the mixture of 250 mL water and 250 mL acetonitrile. The substrate solution was added to the buffer. The reaction volume was filled with water to the end volume of 10 Liter (subtracted volume of enzyme solution and hydrogen peroxide). The shaker was turn on with the speed of 150 to 180 rpm. After 5 minutes shaking 100g cotton fabric was put into the mixture. After that 9 mg Cc/UPO dissolved in 500mL water was added (end concentration of enzyme 0.9 μg/mL). The reaction was started with addition of first portion of hydrogen peroxide (250 mL; 1.25 mM). Hydrogen peroxide was added as 1 L of 50 mM solution in four portion, 250 mL every 30 minutes. After 2 hours, the cotton fabric was removed from the reactor, washed under running water and air-dried.

The originally white fabric turns its color to purple (color intermediate between red and blue); the color is identical to that of Tyrian purple (also known as imperial purple), a natural dye that is prepared from sea snails of the family Muricidae. Example 9: In situ dyeing process based on the conversion of 6-bromoindole to Tyrian purple catalyzed by Agrocybe aegerita peroxygenase

6-bromoindole (1 mM, dissolved in 50/50 v/v acetonitrile/water (ACN/H2O), ACN final concentration was reduced to 2.5%) was converted by 0.025 μg/mL Agrocybe aegerita peroxygenase (/AaeUPO) in 5 L of 50 mM potassium phosphate buffer (pH 7). Total reaction volume was 10 Liter. The same reactor was used as in example 8 (steel tank with a volume of 25 L). The reactor was placed on a laboratory shaker. Prior to hydrogen peroxide addition that initiated the dyeing reaction, about 200 g cotton fabric (two white T-shirts) were added to the reaction mixture. The reaction was started by the addition of a first portion of hydrogen peroxide (1 .25 mM, then continued to 5 mM H2O2 final concentration added in four portion every half an hour). The reactor was shaken with a rotation speed of 180 rpm. After 2 hours, the cotton fabric was removed from the reactor, washed under running water and air-dried. The originally white fabric turns its color to purple (color intermediate between red and blue); the color is identical to that of Tyrian purple (also known as imperial purple), a natural dye that is prepared from sea snails of the family Muricidae. The experimental process was analogous to example 8.

Claims

1. A method for dyeing a textile, comprising: contacting the textile with a peroxygenase, a source of hydrogen peroxide, and an indole, wherein the indole is unsubstituted or substituted once or twice in the benzene ring and wherein each substituent is independently selected from the group consisting of F, CI, Br, OH, NH₂, COOH, S0₃ ^", alkyl and alkoxy.

2. The method of claim 1 , wherein the peroxygenase has an increased selectivity for 3-hydroxy- 1 /-/-indole; preferably the peroxygenase has at least 1 10%, for example, at least 150%, at least 200%, at least 300%, at least 500%, at least 800%, or at least 1000% selectivity for 3-hydroxy- 1 /-/-indole, compared with Agrocybe aegerita unspecific peroxygenase, when the peroxygenase is used to convert the indole to an indigo dye.

3. The method of claim 1 or 2, wherein the peroxygenase comprises or consists of an amino acid sequence which has at least 70% identity, e.g., at least 75% identity, or at least 80% identity, preferably at least 85% identity, more preferably at least 90% identity, further more preferably at least 95% identity, most preferably at least 97% identity, and in particular at least 99% identity to the mature polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, or 18, 19 or 20.

4. The method of any of claims 1-3, wherein the peroxygenase is a peroxygenase variant, comprising an alteration at one or more positions corresponding to positions 5, 6, 1 1 , 19, 26, 28, 34, 39, 44, 52, 54, 55, 58, 59, 62, 65, 71 , 78, 83, 85, 86, 106, 107, 1 17, 120, 147, 151 , 152, 154, 156, 158, 162, 163, 165, 166, 167, 179, 183, 184, 195, 200, 201 , 206, 209, 215 and 216 of the mature polypeptide of SEQ ID NO: 19, wherein each alteration is independently a substitution, deletion or insertion and the variant has peroxygenase activity and wherein the variant has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably the mature polypeptide of SEQ ID NO: 1 , 2, 19 or 20.

5. The method of claim 4, wherein the variant comprises one or more substitutions selected from the group consisting of

W5F

E6K,C

K1 1 N,C

M19L,G H26N,Q,A

F28Y,L,V

R34K

K39Q

G44A

N52Q

T54A

A55L,V,F,I

A58L,V,F,S,I

L59I

F62V,L,A,G

M65T.L

N71 Q

N78D

H83N

I85F,L,V

L86F

N106Q

K107C

T108A

W117F

S120G

F147T

M148L

L151 G,F,A,V

G152A.Q

N153D

I154V,T,A

F155I.V

T156C

G158A,T,F,L

E159D

A162F,L,I,V

Y163D.L

M165I.L

L166V

I167L W179F

W183I.V.F

F184I.V.L

W195F

K200E

E201 S

V206H

A209T

Q215R, and

N216Q.

6. The method of any of claims 4-5, wherein the variant comprises one or more substitutions selected from the group consisting of

A55L

N71 Q

1154V

W183I

W183V

E6K+K1 1 N+R34K+K39Q+G44A+A58S+M65T+N78D+S120G+F147T+N153D+M16 5I+L166V+I167L+K200E+E201 S+V206H+A209T+Q215R

G158A+W183V

G158A+W183I

L151A+G158A

L151A+W183V

K107C+T108A

I85V+M165L

M165L+W179F

W117F+W195F

E6C+W1 17F; and

L151A+W183I.

7. A method for converting a substituted or unsubstituted indole to the corresponding 2,3-epoxy- 1 /-/-indole or 3-hydroxy-1 /-/-indole, comprising contacting the indole with a peroxygenase having an increased selectivity for 3-hydroxy-1 /-/-indole, and a source of hydrogen peroxide; wherein the indole, 2, 3-epoxy-1 /-/-indole, or 3-hydroxy-1 /-/-indole is substituted once or twice in the benzene ring and wherein each substituent is independently selected from the group consisting of F, CI, Br, OH, NH₂, COOH, S0₃ ^", alkyl and alkoxy.

8. A method for preparing a substituted or unsubstituted indigo dye, comprising contacting an indole with a peroxygenase having an increased selectivity for 3-hydroxy-1 /-/-indole, and a source of hydrogen peroxide; wherein the indole is unsubstituted or substituted once or twice in the benzene ring and wherein each substituent is independently selected from the group consisting of F, CI, Br, OH, NH₂, COOH, S0₃ ^", alkyl and alkoxy.

9. The method of claim 7 or 8, wherein the peroxygenase comprises or consists of an amino acid sequence which has at least 70% identity, e.g., at least 75% identity, or at least 80% identity, preferably at least 85% identity, more preferably at least 90% identity, further more preferably at least 95% identity, most preferably at least 97% identity, and in particular at least 99% identity to the mature polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, or 18, 19 or 20. 10. The method of any of claims 7-9, wherein the peroxygenase is a peroxygenase variant, comprising an alteration at one or more positions corresponding to positions 5, 6, 1 1 , 19, 26, 28, 34, 39, 44, 52, 54, 55, 58, 59, 62, 65, 71 , 78, 83, 85, 86, 106, 107, 1 17, 120, 147, 151 , 152, 154, 156, 158, 162, 163, 165, 166, 167, 179, 183, 184, 195, 200, 201 , 206, 209, 215 and 216 of the mature polypeptide of SEQ ID NO: 19, wherein each alteration is independently a substitution, deletion or insertion and the variant has peroxygenase activity and wherein the variant has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9,

10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably the mature polypeptide of SEQ ID NO: 1 , 2, 19 or 20.

1 1. The method of claims 10, wherein the variant comprises one or more substitutions selected from the group consisting of

W5F

E6K,C

K1 1 N,C

M19L,G

H26N,Q,A

F28Y,L,V

R34K

K39Q

G44A

N52Q T54A A55L,V,F,I A58L,V,F,S,I L59I

F62V,L,A,G

M65T.L

N71 Q

N78D

H83N

I85F,L,V

L86F

N106Q

K107C

T108A

W117F

S120G

F147T

M148L

L151 G,F,A,V

G152A.Q

N153D

I154V,T,A

F155I.V

T156C

G158A,T,F,L

E159D

A162F,L,I,V

Y163D.L

M165I.L

L166V

I167L

W179F

W183I,V,F

F184I.V.L

W195F

K200E

E201 S V206H

A209T

Q215R, and

N216Q.

12. The method of claim 10 or 11 , wherein the variant comprises one or more substitutions selected from the group consisting of

A55L

N71 Q

1154V

W183I

W183V

G158A+W183V

G158A+W183I

L151A+G158A

L151A+W183V

K107C+T108A

I85V+M165L

M165L+W179F

W117F+W195F

E6C+W1 17F; and

L151A+W183I.

13. Use of a peroxygenase, a source of hydrogen peroxide, and an indole for in situ dyeing of a textile, wherein the indole is unsubstituted or substituted once or twice in the benzene ring and wherein each substituent is independently selected from the group consisting of F, CI, Br, OH, NH₂, COOH, S0₃ ^", alkyl and alkoxy.

14. Use of a peroxygenase having an increased selectivity for 3-hydroxy-1 /-/-indole for preparing an indigo dye, 2, 3-epoxy-1 /-/-indole or 3-hydroxy-1 /-/-indole, which is unsubstituted or substituted once or twice in the benzene ring(s), and wherein each substituent is independently selected from the group consisting of F, CI, Br, OH, NH₂, COOH, S0₃ ^", alkyl and alkoxy.

15. A peroxygenase variant, comprising an alteration at one or more positions corresponding to positions 5, 6, 1 1 , 19, 26, 28, 34, 39, 44, 52, 54, 55, 58, 59, 62, 65, 71 , 78, 83, 85, 86, 106, 107, 1 17, 120, 147, 151 , 152, 154, 156, 158, 162, 163, 165, 166, 167, 179, 183, 184, 195, 200, 201 , 206, 209, 215 and 216 of the mature polypeptide of SEQ ID NO: 19, wherein each alteration is independently a substitution, deletion or insertion and the variant has peroxygenase activity and wherein the variant has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably the mature polypeptide of SEQ ID NO: 1 , 2, 19 or 20.

16. The peroxygenase variant of claims 15, wherein the variant comprises one or more substitutions selected from the group consisting of

W5F

E6K,C

K1 1 N,C

M19L,G

H26N,Q,A

F28Y,L,V

R34K

K39Q

G44A

N52Q

T54A

A55L,V,F,I

A58L,V,F,S,I

L59I

F62V,L,A,G

M65T,L

N71 Q

N78D

H83N

I85F,L,V

L86F

N106Q

K107C

T108A

W117F

S120G

F147T L151 G,F,A,V

G152A.Q

N153D

I154V,T,A

F155I.V

T156C

G158A,T,F,L

E159D

A162F,L,I,V

Y163D.L

M165I.L

L166V

I167L

W179F

W183I.V.F

F184I.V.L

W195F

K200E

E201 S

V206H

A209T

Q215R, and

N216Q.

17. The peroxygenase variant of claim 15 or 16, wherein the variant comprises one or more substitutions selected from the group consisting of

A55L

N71 Q

1154V

W183I

W183V

G158A+W183V

G158A+W183I

L151A+G158A L151A+W183V K107C+T108A I85V+M165L M165L+W179F W117F+W195F E6C+W117F; and L151A+W183I.