WO2014122109A1

WO2014122109A1 - Enzymatic preparation of indigo dyes and intermediates

Info

Publication number: WO2014122109A1
Application number: PCT/EP2014/052103
Authority: WO
Inventors: Lisbeth Kalum; Henrik Lund; Martin Hofrichter; René ULLRICH
Original assignee: Novozymes A/S
Priority date: 2013-02-05
Filing date: 2014-02-04
Publication date: 2014-08-14

Abstract

The invention relates to enzymatic methods for preparing indigo and derivatives thereof, using peroxygenase and hydrogen peroxide.

Description

ENZYMATIC PREPARATION OF INDIGO DYES AND INTERMEDIATES

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 peroxygenases for preparing indigo dyes, based on indole, or derivatives thereof.

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. It 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.

The chemistry used for producing indigo is quite harsh and not very environmentally friendly. The present invention provides an environmentally friendly alternative for producing indigo by using enzymes. It does not require any harsh reaction conditions, like high

temperatures or highly acid/alkaline pH. SUMMARY OF THE INVENTION

The inventors of the present invention have surprisingly found that peroxygenases can be used to convert indole, and derivates thereof, to the corresponding indigo dyes and/or the intermediary compounds thereof.

Thus, in a first 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 and 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 and 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 yet another aspect, the invention provides a dye composition, comprising an indigo dye and 3,3-dihydro-1 /-/-indol-2-one, which are both 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.

In embodiments, the peroxygenase comprises an amino acid sequence which has at least 60% identity to SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, or 18. 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 H₂0₂ 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%, and even most preferably at least 100% 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, or 18.

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 3.0.0 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)

For purposes of the present invention, the degree of identity between two

deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm

(Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra; http://emboss.org), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) 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 Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in

Alignment).

Modification: The term "modification" means herein any chemical modification of the polypeptide consisting 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, or 18; or a homologous sequence thereof; as well as genetic

manipulation of the DNA encoding such a polypeptide. The modification can be a substitution, a deletion and/or an insertion of one or more (several) amino acids as well as replacements of one or more (several) amino acid side chains.

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.

DETAILED DESCRIPTION OF THE INVENTION

The inventors of the present invention have found that 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 H-indoles; (2) 2,3-epoxy-1 H-indoles; (3) 3,3-dihydro-1 H-indol-2-ones; (4) 3-hydroxy-1 H- 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), 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 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 amino acid sequence of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, or 18; preferably SEQ ID NO:1 .

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: 19).

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

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 ei a/., 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 ei a/., 1988, DNA 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 of interest, and can be applied to polypeptides of unknown structure.

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, or 18;

preferably SEQ ID NO: 1 ; 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 0.05 mg/ml to 50 mg/ml, preferably 0.05 mg/ml to 10 mg/ml, more preferably 0.1 mg/ml to 10 mg/ml, and most preferably 0.1 mg/ml to 5 mg/ml. 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 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 2 mM to 8 mM hydrogen peroxide.

Methods, Compositions and Uses

In a first 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 and hydrogen peroxide; wherein the indole, 2,3-epoxy-1 /-/-indole, or 3-hydroxy-1 /-/-indole (or 3,3-dihydro-1 H- 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, S0₃ ^~, alkyl and alkoxy.

In a second aspect, the invention provides a method for preparing a substituted or unsubstituted indigo dye, comprising contacting an indole with a peroxygenase and 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. In an embodiment, the method is carried out in the presence of oxygen.

In embodiments of the first and second aspects, the peroxygenase comprises an amino acid sequence which has at least 80% identity to SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, or 18. In preferred embodiments, the peroxygenase comprises or consists of an amino acid sequence having at least 85% identity, 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 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, or 18. In more preferred embodiments, the peroxygenase comprises or consists of an amino acid sequence having 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 or SEQ ID NO: 2. In most preferred embodiments, the peroxygenase comprises or consists of the amino acid sequence of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, or 18; or a fragment thereof having peroxygenase activity.

In other embodiments of the first and second aspects, the amino acid sequence of the peroxygenase comprises the motif: E-H-D-[G,A]-S-[L,I]-S-R (SEQ ID NO: 19).

In yet other embodiments of the first and second aspects, each substituent is

independently selected from the group consisting of CI, Br, OH, NH₂, COOH, and S0₃ ^~;

preferably CI, Br, OH, NH₂, and S0₃ ^~; more preferably CI and Br.

In a third aspect, the invention provides a dye composition, comprising an indigo dye and 3,3-dihydro-1 /-/-indol-2-one, which are both 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.

In an embodiment, the composition further comprises indole, which is unsubstituted or substituted with the same substituents in the benzene ring as the indigo dye and 3,3-dihydro- 1 /-/-indol-2-one.

In another embodiment, each substituent is independently selected from the group consisting of CI, Br, OH, NH₂, COOH, and S0₃ ^"; preferably CI, Br, OH, NH₂, and S0₃ ^"; more preferably CI and Br.

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

0₂ is needed, additional oxygen may be added, e.g. as pressurized atmospheric air or as pure pressurized 0₂. 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 the methods and compositions mentioned above for preparing an indigo dye, 2, 3-epoxy-1 /-/-indole or 3-hydroxy-1 /-/-indole (or 3,3-dihydro-1 /-/-indol-2- one), 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.

In an embodiment, each substituent is independently selected from the group consisting of CI, Br, OH, NH₂, COOH, and S0₃ ^~. Preferably, each substituent is independently selected from the group consisting of CI, Br, OH, NH₂, and S0₃ ^~; 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 methods 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, more preferably from 5 minutes to 30 minutes, most preferably from 5 minutes to 20 minutes, and in particular from 5 minutes to 15 minutes.

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

EXAMPLES

Chemicals used as buffers and substrates were commercial products of at least reagent grade. The amino acid sequence of the AaeAPO peroxygenase from Agrocybe aegerita is shown as SEQ ID NO: 1 . The amino acid sequence of the Cc/ΆΡΟ peroxygenase from

Coprinopsis cinerea is shown as SEQ ID NO: 2.

EXAMPLE 1

Conversion of indole and its derivates to the corresponding indigos by peroxygenase Experimental setup

2.5 mM substrate (4-chloroindole, 5-chloroindole, 6-chloroindole, 5-bromoindole, 6- bromoindole, indole) were converted by 7.1 μg mL AaeAPO in 250 μΙ_ of 50 mM potassium phosphate buffer (pH 7) and 20% (v/v) AcN. Hydrogen peroxide was added via a syringe pump at 41 .66 μΜ/min for 60 min (that gives a total concentration of 2.5 mM).

Precipitation of indigo-like dyes was observed in each mixture. The kind and intensity of color was dependent on the substitution pattern of the substrate. Differences were also observed in the amount of dye (semi-quantitative): less dye was formed with unsubstituted indole.

Quantification of all reaction intermediates/products was only done for unsubstituted indole: 2.5 mM of indole was converted into 2.2 mM 3,3-dihydro-1 /-/-indol-2-ones (2-oxindol), traces (<0.1 mM) of isatin, unknown products and precipitated indigo.

EXAMPLE 2

Conversion of 6-chloroindole to 6,6'-dichloroindigo using Agrocybe peroxygenase

2.5 mM 6-chloroindole was converted by 0.284 μg/mL AaeAPO in 25 mL of 50 mM potassium phosphate buffer (pH 7) and 20% (v/v) AcN. H₂0₂ (2.5 mM final concentration) was added via a syringe pump at 41.66 μΜ/min for 60 min (to give a total concentration of 2.5 mM). The reaction mixture was then centrifuged (10 min, at 5,000 g), the precipitated dye was removed (and stored), fresh substrate (ad 2.5 mM) was added and H₂0₂ addition was continued (2.5 mM over 60 min at 41.66 μΜ/min). The produced dye (6,6'-dichloroindigo) was removed again through centrifugation and the reaction was started again by addition of fresh substrate and peroxide.

After the third turn, the reaction mixture was deep brownish-yellow in color and smelled of 6-chloroindole (the substrate), and led us assume that no enzyme activity may have remained. The precipitated dye was collected again, combined with the other dye fractions, dried and weighed. The resulting mass of 1.05 mg means a yield of 9.1 %.

EXAMPLE 3

Conversion of 6-bromoindole to 6,6'-dibromoindigo using Coprinopsis peroxygenase

20 mg 6-bromoindole (approx. 10 mmol) was converted by 0.3 μg/mL Cc/APO in 5 mL of 50 mM potassium phosphate buffer (pH 7) with 20% (v/v) AcN (all concentrations given are final concentrations) and in the presence of 10 mmol H₂0₂. 6-Bromoindole and peroxide were dissolved in AcN/H₂0 (50%/50%, v/v) and supplied to the reaction-vial via a syringe pump over 60 minutes.

The precipitated dye (6,6'-dibromoindigo) was collected, dried and weighed (4.1 mg, i.e. a yield of about 20%). Furthermore, the dried dye sample was sent to Madison (University of Wisconsin) for NMR analysis (to confirm the structure of 6,6'-dibromoindigo).

EXAMPLE 4

Conversion of 6-bromoindole to 6,6'-dibromoindigo using Agrocybe peroxygenase

40 mg 6-bromoindole (approx. 20 mmol) was converted by 1 .775 μg/mL AaeAPO in 5 mL of 50 mM potassium phosphate buffer (pH 7) with 20% AcN (all final concentrations) and 25 mmol H₂0₂. 6-Bromoindole and peroxide were dissolved in AcN/H₂0 (50%/50%, v/v) and supplied via a syringe pump over 360 minutes. The precipitated dye (6,6'-dibromoindigo) was collected, dried and weighed (5 mg, i.e. a yield of ca. 12.5%).

EXAMPLE 5

Conversion of indole to indigo using Agrocybe peroxygenase

50 mg indole (approx. 42 mmol) was converted by 1 .775 μg mL AaeAPO in 5 mL of 50 mM potassium phosphate buffer (pH 7) with 20% (v/v) AcN (all final concentrations) and 50 mmol H₂0₂. Indole and peroxide were dissolved in AcN/H₂0 (50%/50%, v/v) and supplied via a syringe pump over 60 minutes. The precipitated dye was collected by filtration, dried and weighed (0.75 mg, i.e. a yield of about 1 .5%). The identification of the dye product as true indigo was not possible due to a dark green color appearing during the reaction.

Claims

1 . 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 and 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 and wherein each substituent is independently selected from the group consisting of F, CI, Br, OH, NH₂, COOH, S0₃ ^~, alkyl and alkoxy.

2. A method for preparing a substituted or unsubstituted indigo dye, comprising contacting an indole with a peroxygenase and 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.

3. The method of any of claims 1 -2, wherein the peroxygenase comprises an amino acid sequence which has at least 80% identity to SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, or 18.

4. The method of any of claims 1 -3, wherein the amino acid sequence of the peroxygenase comprises the motif: E-H-D-[G,A]-S-[L,I]-S-R (SEQ ID NO: 19).

5. The method of any of claims 1 -4, wherein the peroxygenase comprises or consists of an amino acid sequence having at least 85% identity, 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 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, or 18.

6. The method of any of claims 1 -5, wherein the peroxygenase comprises or consists of an amino acid sequence having 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 or SEQ ID NO: 2.

7. The method of any of claims 1 -6, wherein the peroxygenase comprises or consists of the amino acid sequence of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, or 18; or a fragment thereof having peroxygenase activity.

8. The method of any of claims 1 -7, wherein each substituent is independently selected from the group consisting of CI, Br, OH, NH₂, COOH, and

9. The method of any of claims 1 -8, wherein each substituent is independently selected from the group consisting of CI, Br, OH, NH₂, and S0₃ ^~.

10. The method of any of claims 2-9, which is carried out in the presence of oxygen.

1 1 . A dye composition, comprising an indigo dye and 3,3-dihydro-1 /-/-indol-2-one, which are both 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.

12. The composition of claim 1 1 , which further comprises indole, which is unsubstituted or substituted with the same substituents in the benzene ring as the indigo dye and 3,3-dihydro- 1 H-indol-2-one.

13. The composition of claim 1 1 or 12, wherein each substituent is independently selected from the group consisting of CI, Br, OH, NH₂, COOH, and S0₃ ^~.

14. Use of a peroxygenase 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. The use according to claim 14, wherein each substituent is independently selected from the group consisting of CI, Br, OH, NH₂, and S0₃ ^~.