Photolysis of urine blue

Abstract

The invention relates to application of light to conversion of urine blue into indigo. The invention is particularly useful for dyeing textiles with urine blue by simple light exposure, thereby providing a greener alternative to the current industrial processes of colored fabrics and other products.

Description

Technical Field

[0001] The present invention relates to the use of indican-based light-induced dyeing of textiles, thereby providing a greener alternative to current industrial processes for coloring fabrics and other products. Background Art

[0002] Blue denim is traditionally dyed with chemically synthesized indigo under harsh environmentally challenging conditions. As the final step in the synthesis process, indigo is formed spontaneously from indoxyl by air oxidation, but for dyeing purposes, it is necessary to further dissolve the indigo with a strong reducing agent (e.g., Na2S2O4), which is also an environmentally challenging process ( Figure 1 ).

[0003] Glycosyltransferases (GTs) can be used as a green biotechnology alternative in the current industrial process of blue denim production ( Figure 1 GT glycosylates many different chemicals, including indoxyl (the indigo dye precursor). Specifically, the hydroxyl groups in chemically synthesized indoxyl can be glycosylated by glycosyltransferases, thereby protecting the reactive functional groups and generating a stable, soluble (colorless) indoxyl molecule. Subsequently, the indoxyl can be hydrolyzed back to indoxyl by β-glucosidase (BGL), which can spontaneously oxidize directly on fabric to form blue indigo ( Figure 1 ). This is a “green” alternative to traditional industrial processes, providing an alternative solution for the final steps of the indigo dyeing process, avoiding the use of strong reducing agents.

[0004] The present invention provides a further improvement over the prior art in providing a simpler, greener and cheaper indigo dyeing process. Summary of the invention

[0005] The present invention relates to a method for producing indigo, comprising the following steps:

[0006] a. Providing an aqueous solution containing indican;

[0007] b. Expose the urine blue mother to a light source,

[0008] wherein the urocyanate is exposed to sufficient radiant energy by a light source to convert at least 50% of the urocyanate to indigo; and wherein the glycosidic bonds in the urocyanate are cleaved solely by the light exposure.

[0009] The invention also relates to a method for dyeing a product such as yarn, textile or fabric with indigo, comprising the following steps:

[0010] a. Providing an aqueous solution of urine blue mother;

[0011] b. applying the urine blue mother solution in step (a) to the product; and

[0012] c. exposing the product treated with urine blue masterbatch in step (b) to a light source to obtain a dyed product,

[0013] The glycosidic bonds in the urocyanate are cleaved only by the light exposure.

[0014] In a preferred embodiment, the aqueous solution of step (a) in the process of the present invention is acidic.

[0015] In a preferred embodiment, the wavelength of the light source used to cleave the glycosidic bonds in urocyanate is 300 nm-470 nm.

[0016] In a preferred embodiment, the method of dyeing a product comprises a prior step of glycosyltransferase treatment to glycosylate indoxyl to form urocyanin.

[0017] The invention also relates to the use of light to convert urea blue into indigo.

[0018] Description of the Invention

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 :The traditional process involves chemically synthesized indigo and adding a reducing agent (e.g., sodium dithionite, Na2S2O4) to the indigo vat to reduce it to soluble indigo with dyeing ability. In the "2xenzymatic process", indoxyl is glycosylated at the C3 hydroxyl to form indigo blue as a product. Glycosides (indoxyl) are stable and can be stored. The glucose group is removed from the fabric and during the dyeing step, the regenerated indoxyl is oxidized to indigo on the fabric. No reducing agent is required when dyeing with indigo blue. In the "enzymatic-light process", indoxyl is glycosylated to form indigo blue, which is then exposed to light on the fabric to form indigo. GT = glycosyltransferase, BGL = β-glycosyl hydrolase.

[0021] Figure 2 : Schematic diagram of the structures of the starting, intermediate and final products of the conversion of urocyanin to indigo by the "2x enzymatic method" (top) and the "enzymatic photomethod" (bottom).

[0022] Figure 3: Images of cotton fabric patches that have been dyed with urea blue (top), and their CIELAB values ​​(bottom) for characterization of indigo color. Left: Urea blue hydrolyzed by BGL at pH 9. Right: Urea blue photolysis by LED light.

[0023] Figure 4 : Images of the photolysis of urea blue in solution using (A) a mercury UV lamp, (B) a household light bulb, and (C) an artificial fluorescent bulb as light sources; the different solvents tested are indicated in the figure.

[0024] Figure 5 : UV chromatograms of the photolysis products of urine blue mother at different hydrochloric acid (aq) concentrations. The marked peaks were confirmed to be indigo by NMR.

[0025] Figure 6 : Images of cotton fabric samples dyed once (left) or twice (right) using photolysis of urine blue mother, and their respective CIELAB values ​​for characterizing indigo color, demonstrating that repeated dipping can achieve dynamic coloration similar to the traditional process.

[0026] Figure 7 : Pictures of cotton fabric samples dyed using different mixing modes and their respective CIELAB values ​​for the characterization of indigo color.

[0027] Figure 8 : Pictures of cotton fabric samples dyed at different exposure times and distances between the lamp and the fabric, and the corresponding CIELAB values ​​for indigo color characterization.

[0028] Fig. 9 : Indigo is formed from urea blue by LEDs with different wavelengths.

[0029] Fig.10 : Pictures of cotton fabric samples treated according to the reaction conditions specified in Table 10.

[0030] Fig.11 : Dyeing of other textile materials. (A) STEELOUT sample (90 / 10 wool / nylon), (B) DIVINA (100% wool).

[0031] Fig.12 : Examples of yarn coloring (in duplicate). Conditions: ultrapure aqueous solution of 7 mg / mL urea blue mother, LED light exposure for 3 hours, distance between light and sample 7 cm, (A) immersion for 3 hours, (B) immersion for 6 hours, (C) sample (A) repeated twice.

[0032] Fig.13 : Uroin formation by glycosyltransferase PtUGT1 mut87.

[0033] Abbreviations, Terms and Definitions:

[0034] 'Photolysis' refers to the cleavage of chemical bonds by photolysis, a chemical reaction in which a compound decomposes after absorbing a photon of light.

[0035] The 'light' of the present application is not limited to visible light, but may also include a wider wavelength range, such as the visible spectrum as well as ultraviolet (UV) and infrared (IR).

[0036] 'Radiant flux' or 'radiant power' is the amount of radiant energy (light) emitted, reflected, transmitted or received per unit time. The SI unit of radiant flux is the Watt (W), which is 1 Joule per second (J / s).

[0037] 'Irradiance' is the radiant flux received by a surface per unit area. The SI unit of irradiance is Watt per square meter (W / m 2 ). Irradiance can also be called intensity.

[0038] 'Radiant exposure' is the amount of radiant energy (light) received per unit area of ​​a surface, or equivalently, the cumulative irradiance of a surface over exposure time. The SI unit of radiant exposure is the joule per square meter (J / m 2 ). 'LED' is a light emitting diode. 'LED lamp' or 'LED bulb' refers to an electric lamp that uses LEDs to produce light. LEDs provide efficient lighting in a variety of desired wavelengths.

[0039] An 'OLED' is an organic light emitting diode, also known as an organic electroluminescent (organic EL) diode, in which the light-emitting electroluminescent layer is a thin film of organic compounds that emits light in response to an electric current.

[0040] A 'dye precursor' is defined herein as a compound which, after one or more chemical transformations, yields a dyeing material.

[0041] Glycosyltransferases (GTs) are enzymes that catalyze the formation of glycosidic linkages.

[0042] A 'glycosidic bond' or 'glycoside chain' is a covalent bond that connects one carbohydrate (sugar) molecule to another group. In the present invention, urocyanin contains such a glycosidic bond.

[0043] 'Nucleotide sugar' is defined herein as a molecule in which a sugar is linked to a nucleotide via a glycosidic bond; wherein the sugar is a monosaccharide such as glucose, rhamnose, xylose, arabinose. Nucleotide sugars serve as glycosyl donors in glycosylation reactions; these reactions are catalyzed by glycosyltransferases.

[0044] 'Amino acid sequence identity': The term "sequence identity" as used herein refers to a quantitative measure of the degree of similarity between two amino acid sequences of substantially equal length. The two sequences to be compared must be aligned by inserting gaps or truncating at the ends of the protein sequences to obtain the best match. Sequence identity can be calculated as ((Nref-Ndif)100) / (Nref), where Ndif is the total number of non-identical residues after alignment of the two sequences and Nref is the number of residues in one sequence. Sequence identity calculations are preferably performed automatically using a BLAST program, such as the BLASTP program (Pearson WR and DJ Lipman (1988)) (www.ncbi.nlm.nih.gov / cgi-bin / BLAST). Sequence alignment can be performed using the MAFFT24 program (Multiple Alignment Based on Fast Fourier Transform; Katoh et al. 2019) using default parameters (scoring matrix: blosum 62, gap open penalty: 1.53, gap extension penalty: 0.123).

[0045] Preferably, the number of substitutions, insertions, additions or deletions of one or more amino acid residues in the polypeptide is limited to no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 insertions, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additions, and no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 deletions compared to a control polypeptide. Preferably, these substitutions are conservative amino acid substitutions: limited to replacements between members within the following groups: Group 1: glycine, alanine, valine, leucine, isoleucine; Group 2: serine, cysteine, selenocysteine, threonine, methionine; Group 3: proline; Group 4: phenylalanine, tyrosine, tryptophan; Group 5: aspartic acid, glutamic acid, asparagine, glutamine; Group 6: histidine, lysine, arginine. DETAILED DESCRIPTION

[0046] The present invention relates to the use of urea blue mother to convert indigo using light, which is particularly suitable for dyeing textiles such as twill (demin). Urea blue mother is a glycoside of the indigo precursor indoxyl. Indoxyl compounds can spontaneously dimerize under aerobic conditions to form colored compounds, which can be used as dyes, such as for dyeing fabrics or other products. The dimerized colored compound is insoluble in aqueous solution. Glycosylation of indoxyl stabilizes the compound to prevent its dimerization, thereby providing a soluble dye precursor urea blue mother.

[0047] Indigo has been used to dye denim by enzymatic hydrolysis to break the glycosidic bonds, followed by a spontaneous reaction under aerobic conditions to form indigo. Figure 1The present invention provides a method that uses light instead of enzymes or chemicals, making the process simpler, more environmentally friendly, less expensive and more accessible than prior known technologies ( Figure 1 ).

[0048] Regardless of the route taken – traditional chemical, 2x enzymatic or photoenzymatic – the final compound that produces the blue denim colour is the same chemical entity, indigo ( Figure 1 ).

[0049] I. Urine blue mother into indigo

[0050] The present inventors have discovered that urocyanate can be converted to indigo by simple light exposure. The present invention provides a method for converting urocyanate to indigo by light exposure. Without being bound by theory, it is speculated that the light source converts urocyanate (in solution) into indoxyl radicals by photolysis, and the indoxyl radicals further spontaneously dimerize to form indigo ( Figure 2 ).

[0051] In one aspect, the present invention provides a method for converting urea blue to indigo, comprising the steps of:

[0052] a. Providing an aqueous solution of urine blue mother;

[0053] b. Expose the urine blue mother to light,

[0054] wherein the conversion is mediated solely by said light exposure.

[0055] In one embodiment, the present invention provides a method for converting urea blue mother into indigo, comprising the following steps:

[0056] a. Providing an aqueous solution containing urine blue mother;

[0057] b. Expose the urine blue mother to a light source,

[0058] wherein the urocyanate is exposed to sufficient radiation energy by a light source to convert at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or even 90% of the urocyanate into indigo; and wherein the glycosidic bonds in the urocyanate are cleaved solely by said light exposure.

[0059] In a preferred embodiment, the present invention provides a method for converting urea blue mother into indigo, comprising the following steps:

[0060] a. Providing an aqueous solution containing urine blue mother;

[0061] b. Expose the urine blue mother to a light source,

[0062] wherein the urocyanate is exposed to sufficient radiation energy by a light source to convert at least 50% of the urocyanate into indigo; and wherein the glycosidic bonds in the urocyanate are cleaved solely by the light exposure.

[0063] In a preferred embodiment, the present invention provides a method for converting urea blue mother into indigo, comprising the following steps:

[0064] a. Providing an aqueous solution containing urine blue mother;

[0065] b. Expose the urine blue mother to a light source,

[0066] wherein the urocyanate is exposed to sufficient radiation energy by a light source to completely convert the urocyanate to indigo, consuming at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the urocyanate; and wherein the glycosidic bonds in the urocyanate are cleaved solely by said light exposure.

[0067] Light exposure mediates the conversion of urocyanin to indigo by cleaving the glycosidic bonds in the urocyanin. In particular, in a preferred embodiment, the present invention provides a method for converting urocyanin to indigo that does not include the use of β-glycosyl hydrolase (enzyme classification number E.C 3.2.1.21).

[0068] In a preferred embodiment, the conversion of urocyanin to indigo involves the formation of an indoxyl radical as an intermediate product.

[0069] In a preferred embodiment, the conversion of urea blue to indigo includes (i) the formation of indoxyl radical as an intermediate, and (ii) spontaneous dimerization and oxidation to form indigo.

[0070] Ii Light Source

[0071] As demonstrated in the examples, conversion of urea blue to indigo by light exposure is applicable to a range of different light sources, including natural sunlight, artificial sunlight bulbs, and certain LEDs, thereby achieving the traditional blue denim color sought by consumers.

[0072] In some embodiments, the light source may include one or more lasers, LEDs / OLEDs, incandescent lamps, mercury lamps, UV lamps, arc lamps, argon lamps, or any other gas lamps (e.g., neon lamps and krypton lamps) with specific or adjustable wavelengths. In some embodiments, the light source may include natural sunlight or artificial daylight bulbs. The light source may be pulsed (i.e., turned on in bursts for a limited time) or continuous, gated, filtered, amplified, attenuated, polarized, or otherwise regulated by a physical shutter to generate light for photolysis of urocyanin.

[0073] The glycosidic bonds in the urocyanate are excited by a specific wavelength or a range of wavelengths, inducing photocleavage in an efficient manner. In one embodiment, the wavelength of the light source is in the range of 300-470nm, 300-450nm, 310-440nm, 320-430nm, 330-420nm, 340-410nm, 350-400nm, 360-390nm, 370-380nm, such as a wavelength of about 375nm.

[0074] In a preferred embodiment, the wavelength of the light source used to convert urocyanin to indigo - i.e., cleave the glycosidic bonds in urocyanin - is between 300-450nm, 310-440nm, 320-430nm, 330-420nm, 340-410nm, 350-400nm, 360-390nm, 370-380nm.

[0075] In one embodiment, the wavelength of the light source is 300nm, 305nm, 310nm, 315nm, 320nm, 325nm, 330nm, 335nm, 340nm, 345nm, 350nm, 355nm, 360nm, 365nm, 370nm, 375nm, 380nm, 385nm, 390nm, 395nm, 400nm, 405nm, 410nm, 415nm, 420nm, 425nm, 430nm, 435nm, 440nm, 445nm, 450nm, 455nm, 460nm, 465nm, 470nm, 475nm, 480nm, 485nm, 490nm, 495nm, or 500nm.

[0076] II. A method for producing indigo and a method for dyeing the product

[0077] In one aspect, the present invention provides a method for producing indigo, comprising the steps of:

[0078] a. Providing an aqueous solution containing urine blue mother;

[0079] b. Expose the urine blue mother to a light source,

[0080] wherein the urocyanate is exposed to sufficient radiation energy by a light source to convert at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the urocyanate into indigo; wherein the glycosidic bonds in the urocyanate are cleaved only by the light exposure.

[0081] In a further aspect, the present invention provides a method for dyeing a product with indigo, comprising the steps of:

[0082] a. Providing an aqueous solution of urine blue mother;

[0083] b. applying the urine blue mother solution in step (a) to the product; and

[0084] c. exposing the product treated with urine blue masterbatch in step (b) to a light source to obtain a dyed product,

[0085] Wherein, the glycosidic bonds in the urocyanate are cleaved only by the light exposure.

[0086] The process of dyeing a product using indigo may be repeated multiple times - such as 2, 3, 4, 5 or more times - to obtain a darker / darker product color.

[0087] In particular, the present invention provides a method for producing indigo from urea blue, and optionally using the indigo in dyeing a product, wherein the method does not include the use of a β-glycosyl hydrolase (Enzyme Classification Number E.C 3.2.1), such as a β-glucosidase (EC 3.2.1.21).

[0088] In some embodiments, the product for dyeing by the method disclosed herein can be selected from yarn, textile, fabric and similar products. In a preferred embodiment, the product is yarn or textile.

[0089] II.i. Provide urine blue mother

[0090] Indigo is produced from urocyanin in the present invention. The urocyanin can be provided by different methods. In one embodiment, the urocyanin can be obtained from indoxyl by enzymatic glycosylation. The glycosyltransferase has glycosyltransferase activity (enzyme classification EC: 2.4.1.-) for glycosylation of compounds with reactive groups.

[0091] In one embodiment, the urocyanate is provided by the following steps: providing (i) indoxyphenol, (ii) nucleotide sugar and (iii) a polypeptide having glycosyltransferase activity, mixing components (i), (ii) and (iii), preferably under reaction conditions where the free oxygen content is less than 2%, and reacting the mixture to obtain the urocyanate.

[0092] In the present invention, the glycosyltransferase glycosylates indoxyl to form uropyrimidine, which is a soluble, stable indigo dye precursor. In a preferred embodiment, the glycosyltransferase has indoxyl-UDP-glucosyltransferase activity (enzyme classification EC: 2.4.1.220). In one embodiment, the amino acid sequence of the polypeptide having glycosyltransferase activity has at least 75% sequence identity with the UDP-dependent glycosyltransferase from Polygonum / Indigofera (PtUGT1, SEQ ID NO: 2), such as at least 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97% sequence identity with PtUGT1. In a most preferred embodiment, the amino acid sequence of the polypeptide having glycosyltransferase activity is SEQ ID NO: 4. In another most preferred embodiment, the amino acid sequence of the polypeptide having glycosyltransferase activity is SEQ ID NO: 6. In another most preferred embodiment, the amino acid sequence of the polypeptide having glycosyltransferase activity is SEQ ID NO:8.

[0093] The skilled person is familiar with methods for providing enzymes. Such enzymes may be produced, for example, by microorganisms - such as recombinantly or by natural producers, or synthesized.

[0094] In one embodiment, nucleotide sugar is UPD sugar, such as UDP-glucose, UPD-rhamnose, UPD-xylose and UPD-arabinose. In a preferred embodiment, nucleotide sugar is UDP-glucose. UDP-glucose can be directly provided as UDP-glucose, and also can be provided indirectly with the form of other sugars or sugary molecules, and these sugars are converted into UDP-glucose subsequently. Such an example of indirectly providing UPD-glucose is to provide sucrose and UDP, and then it is converted into UDP-glucose by enzyme catalysis (for example using sucrose synthase (SuSy) EC2.4.1.13).

[0095] Indoxyl can react with oxygen in the presence of oxygen - for example competing with enzymatic glycosylation reactions. Therefore, providing an oxygen-reduced, anaerobic or substantially anaerobic environment is beneficial for the glycosylation reaction to ensure that indoxyl does not spontaneously dimerize.

[0096] In one embodiment, the reaction conditions for the glycosylation of indoxyl to form indigo are substantially anaerobic. In one embodiment, the reaction conditions include less than 2% free oxygen, such as less than 1.5%, 1%, 0.5%, or even less than 0.1% free oxygen and / or maintain a pressure of less than 10 kPa, 9 kPa, 8 kPa, 7 kPa, 6 kPa, 5 kPa, 4 kPa, 3 kPa, 2 kPa or even less than 1 kPa to reduce the possibility of oxidation of the active groups of the compound - i.e., to reduce the possibility of spontaneous dimerization of indoxyl to form indigo.

[0097] The indoxyl is preferably incubated with the glycosyltransferase under conditions of temperature and pH that are optimal for the enzyme, which are known and routinely optimized by those skilled in the art.

[0098] II.ii. Aqueous solution containing urocyanin

[0099] The urea blue mother provided in step (a) of the method for producing indigo from urea blue mother and the method for dyeing the product with indigo are carried out in aqueous solution. In a preferred embodiment, the aqueous solution is neutral or acidic. These conditions provide the most complete conversion (clean conversion) of urea blue mother into indigo, with little or no by-product formation. In one embodiment, the pH value of the urea blue mother solution is or is less than 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, or even less than 1.0. In one embodiment, the pH value range of the urea blue mother solution is 1-8, 1-7, 1-6, 1-5, 1-4 or 1-3. In one embodiment, the aqueous solution is an acidic solution of HCl, CH3COOH, CF3COOH, HNO3, HCOOH and H2SO4.

[0100] The concentration of urea blue in the aqueous solution used to dye the product is preferably at least 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 120mM, 140mM, 160mM, 180mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, 500mM, 550mM, 600mM or even 650mM urea blue, such as concentrated The concentration of urine blue mother is between 20-600mM, 20-500mM, 20-400mM, 20-300mM, 20-250mM, 20-200mM, 20-180mM, 20-160mM, 20-140mM, 20-120mM, 20-100mM, 20-90mM, 20-80mM, 20-70mM, 20-60mM, 20-40mM or 30-60mM or 35-50mM.

[0101] The preferred amount of urea blue in the solution depends on the application of the final indigo product. In a preferred embodiment, the acidic aqueous solution containing urea blue is used in a textile dyeing process, wherein the amount of urea blue used in the dyeing process is 0.05-100 μmol / cm 2 , 0.1-50μmol / cm 2 , 0.1-45μmol / cm 2 , 0.1-40μmol / cm 2 , 0.1-35μmol / cm 2 , 0.1-30μmol / cm 2 , 0.1-25μmol / cm 2 , 0.1-20μmol / cm 2 , 0.1-15μmol / cm 2 , 0.1-10μmol / cm 2 , 0.1-9μmol / cm 2 , 0.1-8μmol / cm 2 , 0.1-7μmol / cm 2 , 0.1-6μmol / cm 2 , 0.1-5μmol / cm 2 , 0.1-4μmol / cm 2 , 0.1-3μmol / cm 2 , 0.1-2μmol / cm 2 , 0.1-1μmol / cm 2 fabric, or 0.2-5μmol / cm 2 , 0.3-5μmol / cm 2 , 0.4-5μmol / cm 2 , 0.5-5μmol / cm 2 , 0.6-5μmol / cm 2 , 0.7-5μmol / cm 2 , 0.8-5μmol / cm 2 , 0.9-5μmol / cm 2 , 1-5μmol / cm 2 , 1.5-5μmol / cm 2 , 2-5μmol / cm 2 , 2.5-5μmol / cm 2 , 3-5μmol / cm 2 、3.5-5μmol / cm 2 , 4-5μmol / cm 2 , 5.5-5μmol / cm 2fabric, or 0.5-1.5μmol / cm 2 , 0.3-2μmol / cm 2 , 0.1-4μmol / cm 2 , 0.05-10μmol / cm 2 , or 0.1-10 μmol / cm 2 fabric.

[0102] II.iii. Light exposure

[0103] The light source and preferred wavelength are as described above. The light exposure is preferably carried out under wet conditions - ie the conversion of the urea blue to indigo occurs in aqueous solution.

[0104] In some preferred embodiments, the urea blue master solution is applied by mixing, immersing, soaking or coating the product in the urea blue master solution, and the product is exposed to light in the urea blue master solution. In other words, the urea blue master treated product is preferably not dried before light exposure. In another embodiment, shaking, stirring or other mixing methods are preferably used during light exposure to ensure that the desired ring pattern is formed in the photochemical coloration of the fabric.

[0105] In one embodiment, when the light source is natural sunlight, the urea blue treated product for dyeing is simply placed in natural sunlight, such as outdoors in an open air environment.

[0106] In some embodiments, the product treated with urea blue is exposed to light by bringing a light source close to the fluid structure, whether or not a lens or other optical system is used, wherein the location, light intensity, and duration of exposure are controlled by those skilled in the art. The total light (radiation) exposure of the selected product depends on the light source applied, the distance between the light source and the product, and the total exposure time.

[0107] In one embodiment, the product treated with urea blue is irradiated with a light source, wherein the product receives an average of 20-220 W / m 2 、30-220W / m 2 、40-220W / m 2 、50-220W / m 2 、60-220W / m 2 、70-220W / m 2 、80-220W / m 2 、90-220W / m 2 , 100-220W / m 2 、110-220W / m 2 , 120-220W / m 2 、130-220W / m 2、140-220W / m 2 , 150-220W / m 2 、160-220W / m 2 、170-220W / m 2 、180-220W / m 2 、190-220W / m 2 , 200-220W / m 2 , 210-220W / m 2 or 20-30W / m 2 , 20-40W / m 2 , 20-50W / m 2 , 0-60W / m 2 , 20-70W / m 2 , 20-80W / m 2 , 20-90W / m 2 , 20-100W / m 2 、20-110W / m 2 , 20-120W / m 2 、20-130W / m 2 、20-140W / m 2 , 20-150W / m 2 、20-160W / m 2 、20-170W / m 2 , 20-180W / m 2 、20-190W / m 2 , 20-200W / m 2 、20-210W / m 2 In one embodiment, the product treated with urea blue is irradiated with a light source, wherein the product receives an average of 20-220 W / m 2 、30-210W / m 2 , 40-200W / m 2 、50-190W / m 2 、60-180W / m 2 、70-170W / m 2 、80-160W / m 2 、90-150W / m 2 100-140W / m 2 or 110-130W / m 2 In certain embodiments, the product treated with urea blue is irradiated with a light source, wherein the product receives 37-208 W / m 2 、65-208W / m 2 or 109-168W / m 2 .

[0108] In one embodiment, the urea blue treated product is exposed to the light source for at least 5 minutes, 15 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes, 240 minutes, 270 minutes, 300 minutes or even 360 minutes, such as the exposure period is between 5-60 minutes, 30-120 minutes, 90-240 minutes or 120-360 minutes.

[0109] In one embodiment, the final radiation exposure of the indigo treated product is between 100 and 10,000 J / m 2 , 500-10000J / m 2 1000-10000J / m 2 1500-10000J / m 2 2000-10000J / m 2 , 2500-10000J / m 2 3000-10000J / m 2 、3500-10000J / m 2 4000-10000J / m 2 4500-10000J / m 2 , 5000-10000J / m 2 , 5500-10000J / m 2 、6000-10000J / m 2 、6500-10000J / m 2 , 7000-10000J / m 2 , 7500-10000J / m 2 , 8000-10000J / m 2 、8500-10000J / m 2 , 9000-10000J / m 2 , 9500-10000J / m 2 Between or between 100-500 J / m 2 100-1000J / m 2 100-1500J / m 2 100-2000J / m 2 100-2500J / m 2 100-2500J / m 2 100-3000J / m 2 100-3500J / m 2 100-4000J / m 2 100-4500J / m2 100-5000J / m 2 100-5500J / m 2 100-6000J / m 2 100-6500J / m 2 100-7000J / m 2 100-7500J / m 2 100-8000J / m 2 100-8500J / m 2 100-9000J / m 2 100-9500J / m 2 In some embodiments, the final radiation exposure of the indigo treated product is 100-10000, 250-5000, 500-3000, 600-2000, 700-1500 kJ / m 2 In some embodiments, the final radiation exposure of the indigo treated product is 100-10000 kJ / m 2 、600-1000kJ / m 2 or 1000-2000J / m 2 Too little radiation exposure may result in a product that is too weakly colored, while too much radiation exposure may cause off-coloring, such as by the formation of by-products or may even destroy the indigo that has already been formed.

[0110] II.iv Testing of intermediates and final products

[0111] The intermediates and compounds produced by the present invention can be detected by HLPC-UV, LC-MS, NMR or similar analytical equipment recognized by those skilled in the art.

[0112] The CIELAB color space, also known as L*a*b*, is a color space defined by the International Commission on Illumination (CIE for short). It represents color by three values: L* represents perceived brightness, and a* and b* represent the four unique colors in human vision: red, green, blue, and yellow, respectively. CIELAB is used in industry to detect color differences. CIELAB values ​​are measured using a benchtop spectrophotometer (MetaVueVS3200, X-Rite (USA)). Indigo can be reported by its L*a*b* value.

[0113] II.vIndigo blue color

[0114] In a preferred embodiment, the color of the indigo dye obtained by the method of the present invention is similar to the color of standard blue indigo denim. Preferably, the color of the final indigo dyed product of the present invention has an L* value of 10-80, an a* value of -5-5, and a b* value of -20-0.

[0115] Those skilled in the art will recognize that the different characteristics of the above light sources will affect the color development. The wavelengths disclosed will excite the glycosidic bond in the urea blue mother and cleave the bond, thereby spontaneously forming indigo. The intensity and purity of the color depends, among other things, on the total radiation exposure - which will affect the final color of the dyed product.

[0116] V. The use of light in denim dyeing

[0117] In another aspect, the invention discloses the use of light to convert urea blue to indigo. In preferred embodiments, the wavelength of the light is between 300-450nm, 310-440nm, 320-430nm, 330-420nm, 340-410nm, 350-400nm, 360-390nm, 370-380nm. In further preferred embodiments, the indigo product is simultaneously used to dye a product such as a textile or fabric.

[0118] VI. Advantages and Commercial Applications

[0119] As previously mentioned, and further demonstrated in the following examples, the method of the present invention provides a more environmentally friendly and less costly alternative for current fabric and textile dyeing processes. In fact, using light, such as natural sunlight, to dye denim makes this denim dyeing method both cheap (almost free), sustainable and low-tech. The main advantage is its sustainability, because there is no need to use irritating chemicals or expensive enzymes. In addition, urine blue mother can be directly applied to the fabric before light exposure, so that indigo is only formed on the fabric, thereby reducing the indigo content in the wastewater, which is another major environmental issue. Although most dyeing processes are currently carried out in countries with lower ecological regulations, denim suppliers and users may have a growing demand for more sustainable processes.

[0120] Preferred embodiments of the present invention

[0121] Preferred embodiment 1: A method for producing indigo, comprising the following steps:

[0122] a. providing an aqueous solution containing urine blue mother;

[0123] b. Expose the urine blue mother to a light source,

[0124] wherein the urocyanate is exposed to sufficient radiation energy by a light source to convert at least 50% of the urocyanate into indigo; and wherein the glycosidic bonds in the urocyanate are cleaved solely by the light exposure.

[0125] Preferred embodiment 2: A method for dyeing a product with indigo, comprising the following steps:

[0126] a. Providing an aqueous solution of urine blue mother;

[0127] b. applying the urine blue mother solution in step (a) to the product; and

[0128] c. exposing the product treated with urine blue masterbatch in step (b) to a light source to obtain a dyed product,

[0129] Wherein, the glycosidic bonds in the urocyanate are cleaved only by the light exposure.

[0130] Preferred embodiment 3: The method according to preferred embodiment 1 or 2, wherein the aqueous solution in step (a) is acidic.

[0131] Preferred embodiment 4: The method according to any one of preferred embodiments 1-3, wherein the light source is selected from sunlight, artificial daylight bulbs, broadband light sources, and LED lamps.

[0132] Preferred embodiment 5: The method according to any one of preferred embodiments 1-4, wherein the wavelength of the light source is 300-470nm.

[0133] Preferred embodiment 6: The method according to any one of preferred embodiments 2-5, wherein the product treated with urea blue is illuminated by the light source at 20-220 W / m 2 Irradiation.

[0134] Preferred embodiment 7: The method according to any one of preferred embodiments 2-6, wherein the product treated with urea blue is exposed to the light source for up to 300 minutes.

[0135] Preferred embodiment 8: The method according to any one of preferred embodiments 2-7, wherein the final radiation exposure of the product treated with urea blue is 100-10000 J / m 2 .

[0136] Preferred embodiment 9: The method according to any one of preferred embodiments 2-8, wherein the light exposure is performed while the product is immersed or soaked in the urine blue mother solution.

[0137] Preferred embodiment 10: The method according to any one of the preferred embodiments 2-9, wherein the product is selected from one or more of yarn, textile and fabric, preferably a cotton-based product.

[0138] Preferred embodiment 11: The method according to any one of preferred embodiments 2-10, wherein the product is a textile or fabric, and wherein the concentration of urea blue in the urea blue solution is 100% per cm 2 Textiles or fabrics 0.1-10μmol.

[0139] Preferred embodiment 12: A method according to any one of preferred embodiments 1-11, wherein step (a) comprises: providing (i) indoxyphenol, (ii) nucleotide sugar and (iii) a polypeptide having glycosyltransferase activity, mixing components (i), (ii) and (iii), preferably under reaction conditions where the free oxygen content is less than 2%, and reacting the mixture to obtain urine blue mother.

[0140] Preferred embodiment 13: Use of light to convert urea blue to indigo.

[0141] Preferred embodiment 14: Use of light according to preferred embodiment 13, wherein the indigo is simultaneously used for dyeing products.

[0142] Preferred embodiment 15: Use of light according to preferred embodiment 13 or 14, wherein the wavelength of the light is 300-450 nm.

[0143] Example

[0144] General approach

[0145] CIELAB color space (L*, a* and b*) values ​​were obtained using a benchtop spectrophotometer (MetaVue VS3200, X-Rite (USA)). CIEL data from 60 commercial blue jeans fabric samples are presented in violin plots and box plots, where the values ​​(bullet shapes) of experimental fabric samples of different colors are superimposed in the present invention.

[0146] Example 1: Dyeing denim using sunlight vs enzymes

[0147] Sunlight exposure: 1-5 μmol / cm 2 The urine blue mother was dissolved at 100 μL / cm 2 , added to the culture dish with an area of ​​20cm 2 The denim discs to be dyed (diameter 5.04 cm, weight 798 + / - 2 mg) (kindly provided by NudieJeans, Sweden) were exposed to sunlight-like light for 4 hours at room temperature by using an artificial daylight bulb (OSRAM, UVITALX 300W E) without stirring. The discs were then washed with water and detergent and dried for CIE analysis.

[0148] Enzyme treatment (β-glucosidase): 1-5 μmol / cm 2 The urine blue mother was dissolved at 100 μL / cm 2 , added to the culture dish with an area of ​​20cm 2 (diameter 5.04cm, weight 798+ / -2mg) on ​​a denim disc to be dyed. Then add 2mg / cm 2 ScGlu (Secale cereale beta-glucosidase), dissolved in 50 μL / cm 2 The enzymatic reaction was carried out in 50 mM phosphate-citrate buffer (pH 9, final concentration 16.7 mM) at room temperature, protected from light, and without stirring for 4 hours. Subsequently, the disc was washed with water and detergent, dried, and analyzed by CIE.

[0149] The CEIL values ​​of the dyed textiles are shown in Table 1.

[0150]

[0151]

[0152] Conditions for each B#.

[0153] B31: Urine blue concentration = 1 μmol / cm 2 , volume = 0.23 ml / cm 2 , container cover = yes, mixing source = magnet, exposure time = 6 hours, number of repetitions = 1, lamp distance = 30 cm.

[0154] B35: Urine blue concentration = 6.8 μmol / cm 2 , volume = 0.23 ml / cm 2 , Container Cover = Yes, Mixing Source = Magnetic, Exposure Time = 6 hours, Number of Repetitions = 1, Lamp Distance = 30 cm.

[0155] B36: Urine blue concentration = 1 μmol / cm 2 , volume = 0.23 ml / cm2, container cover = yes, mixing source = magnetic, exposure time = 6 hours, number of repetitions = 2, lamp distance = 30 cm.

[0156] B58C: Urine blue concentration = 1 μmol / cm 2 , volume = 0.45 ml / cm 2 , Container Cover = None, Mixing Source = Shaker, Exposure Time = 2 hrs, Repetitions = 1, Lamp Distance = 55 cm.

[0157] Figure 3Further proof of the feasibility of the formation of indigo from urea blue by photolysis is shown by comparing the pictures of fabric samples dyed by the enzymatic method (sample B36 front) and the pictures of fabric samples dyed by the photolysis process of the present invention (sample BGL2 front) and the pictures of fabric samples dyed by the photolysis process of the present invention (sample BGL2 front) and the corresponding CEIL values. Indeed, the simple photolysis process is able to obtain a satisfactory blue denim color.

[0158] Example 2: Testing different light sources and solvents

[0159] Urine blue mother (5mg, 16.93μmol) was dissolved in a selected solvent (520μL), placed in a glass container, and then sealed with a glass lid. The sealed container was placed under a selected light source for 2 hours, and then LCMS analysis was performed. The solvents tested included: 80mM NaOH (aq), 80mM HCl (aq), ultrapure water (milli-Qwater), dimethyl sulfoxide (DMSO), methanol (MeOH) and acetonitrile (ACN). The light sources tested were: mercury ultraviolet bulbs (Omnilux, 89514005), household bulbs (DURA LAMP, 07018) and artificial daylight bulbs (OSRAM, U VITALX 300W E).

[0160] The results are as follows Figure 4 and Table 2. Interestingly, a range of different colors were observed depending on the solvent used. HCl was found to be a good solvent candidate to obtain the desired blue denim color by photolysis of urea blue to indigo.

[0161]

[0162] To further optimize the purity of the product, different hydrochloric acid concentrations were tested. Product characteristics were measured and analyzed by LCMS using a Waters AQUITY UPLC system equipped with a PDA and SQD electrospray mass spectrometry detector. Column: Thermoaccucore C18 2.6μm, 2.1×50mm. Column temperature: 50°C. Flow rate: 0.6mL / min. Solvent A1-0.1% formic acid in water, solvent B1-0.1% formic acid in ACN. Gradient: from 5% B to 100% B in 3min, hold 0.1min, total run time 5min. Chromatograms are shown in Figure 5 LCMS was used to quantify the consumption of urocyanin.

[0163] Varying the pH between 1 and 2 (using hydrochloric acid as solvent) determined pH 1.7 (20 mM HCl (aq)) to be optimal, such as with respect to minimal by-product formation.

[0164] The peak marked (boxed) was identified by NMR as indigo, and color extracted from commercial denim also produced a peak with the same retention time (data not shown).

[0165] Example 3: Fabric dyeing

[0166] 3.1 Repeated dipping

[0167] Artificial daylight bulbs (simulating natural sunlight) are used for photolysis of urea blue and dyeing of fabrics. Reaction conditions: 1 μmol urea blue / (cm 2 fabric), 0.225 mL of 20 mM HCl(aq) / (cm 2 fabric), magnetic stirring, 6 hours exposure time. Figure 6 (Pictures of dyed fabrics) and Table 3 (CIEL values ​​of dyed fabrics). The pictures and CIELAB values ​​show that repeated dipping can achieve dynamic coloring, similar to the traditional dyeing process.

[0168]

[0169] 3.2 Different hybrid approaches

[0170] Artificial daylight bulbs were used for photolysis of urea blue and dyeing of fabrics. Reaction conditions: 1 μmol urea blue / (cm2 fabric), 2x immersion in 20 mM HC(aq), exposure time 6 hours. Results are shown in Figure 7 (Pictures of dyed fabrics) and Table 4 (CIEL values ​​of dyed fabrics).

[0171]

[0172] The data showed that the mixing mode affected the color development, and the magnetic force led to uneven coloration, where the distribution depended on the location of the magnetic force. No mixing resulted in lighter colors, i.e., less efficient use of the urine blue masterbatch. Shaking produced uniform and efficient color development, but promoted the formation of byproducts (red, probably due to over-ventilation), which could be alleviated by increasing the liquid volume, expressed here as the amount of liquid per square centimeter of fabric.

[0173] 3.3 Exposure time and illumination area

[0174] Artificial daylight bulbs are used for photolysis of urea blue and dyeing of fabrics. Reaction conditions: 1 μmol urea blue / (cm 2 fabric), 0.45 mL of 20 mM HCl(aq) / (cm 2 fabric), shake for mixing method, 2x dip. Results are shown in Figure 8 (Picture of dyed fabric) and Table 5 (CIEL values ​​of dyed fabric).

[0175]

[0176] The data showed that reducing the exposure time in half and increasing the lamp distance to allow a larger area to be illuminated did not affect color quality.

[0177] Example 4: Wavelength Optimization

[0178] Testing 10 LEDs at different wavelengths allowed the identification of the optimal wavelength for photolytic cleavage of urocyanin.

[0179] To determine which specific wavelengths are responsible for initiating the photolysis process, a series of 10 LEDs with specific wavelengths were tested, these wavelengths are 308nm, 325nm, 340nm, 365nm, 415nm, 470nm, 530nm, 590nm, 625nm and 660nm. These LEDs were purchased from Thorlabs, 308nm (M310L1), 325nm (M325L5), 340nm (M340L4), 365nm (M365L2), 415nm (M415L4), 470nm (M470L5), 530nm (M530L4), 590nm (M590L4), 625nm (M625L4) and 660nm (M660L4).

[0180] Specifically, 5 mg of urocyanin was dissolved in 520 μL of 20 mM HCl (aq), which was placed in a glass container covered with a quartz glass lid and placed 14 cm below the LED. The sample was exposed to light for 2 hours before LCMS analysis. The study found that the 365 nm wavelength gave the highest conversion of urocyanin to indigo; the results are shown in Fig. 9 .

[0181] Example 5: Using a collimator

[0182] The photolysis reaction was further optimized using the optimal wavelength determined above. The light source was a single diode (365 nm) modified from an Olympus BX and IX equipped with a collimator (50 mm 2 )(Olympus BX&IX+365nm LED(M660L4-C1)).

[0183] The reaction conditions and results are shown in Tables 10 and Fig.10 .

[0184]

[0185] 1 The irradiance was measured using an integrating sphere spectroradiometer (Instrument Systems, ISP150 and CAS140D).

[0186] Example 6: Other fabric materials

[0187] 90 / 10 wool / nylon (STEELOUT) and 100% wool (DIVINA) samples were dyed following the procedures described in Examples 3 and 4, respectively.

[0188] Specifically, 2.46 μmol of urinary blue mother / (cm 2 Fabric) was dissolved in 1.13 mL of 20 mM HCl (aq) / (cm 2 fabric), placed in a glass container slightly wider than the diameter of the fabric and placed 7 cm below the LED. The samples were exposed to light for 3 hours; the results are shown in Fig.10 (A: STEELOUT front and B: DIVINA front).

[0189] Example 7: Yarn dyeing

[0190] The procedures of Examples 3 and 4 were redesigned for dyeing yarn samples. A solution of urea blue was prepared in a dark glass container and kept in a dark box at all times. The yarn was cut into 15 cm sample size and soaked in the above solution, then transferred to a glass container containing 20 mM HCl and exposed to LED (365 nm). Different conditions were tested; (1) the solvent of the soaking solution (20 mM HCl or ultrapure water), (2) the concentration of urea blue in the soaking solution (7 mg / mL, 5 mg / mL, 3 mg / mL, 2 mg / mL, 1 mg / mL), (3) soaking time (1 hour, 2 hours, 3 hours, 6 hours), (4) the time of light exposure (1 hour, 2 hours, 3 hours) and (5) the number of repetitions (1 time, 2 times, 3 times).

[0191] A specific example: Under dark conditions, urea blue mother (5 mg / mL) was dissolved in ultrapure water (5 mL) to prepare the soaking solution. Two yarn (15 cm) samples were soaked in the solution for 3 hours, then transferred to a glass container (4 cm diameter) containing 20 mM HCl (4 mL) and exposed to LED (365 nm) for 3 hours. When the experiment was repeated multiple times, the yarn was rinsed before returning to the soaking solution container, and the rest of the steps were the same as above. Fig.12 Three embodiments are shown.

[0192] Example 6: Production of urocyanin by glycosylation of indoxyl

[0193] The synthesis of urocyanin from indoxyl acetate by glycosyltransferase (GT) was demonstrated using a glycosyltransferase having SEQ ID NO: 4 (PtUGT1 mut87).

[0194] Urocyanine was synthesized from high concentrations of indoleacetic acid (100 mM) in an anaerobic chamber using glass HPLC vials, stirred with a small magnetic stirrer, and performed in triplicate at 30°C. The reaction system included 3.5 mg of indoleacetic acid, 90 mM phosphate-citrate buffer (pH 8), 1 mM UDP, 200 mM sucrose, 2 U esterase from Bacillus subtilis (Sigma Aldrich), and different concentrations of GT / SuSy, always at a molar ratio of 1:5 (GT 50 μg, 20 μg, 10 μg, 5 μg; SuSy 432.5 μg, 173 μg, 86.7 μg, 43.3 μg). Sucrose synthase (SuSy) converts sucrose and uridine 5'-diphosphate (UDP) into UDP-glucose. The reaction was initiated by adding all three enzymes (esterase, GT, and SuSy), and the progress of the reaction was monitored by HPLC. Samples were collected at 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours and 32 hours.

[0195] Fig.13 The GT enzyme was shown to be efficient in producing urocyanin from indoleacetate.

Claims

1. A method for producing indigo, comprising the following steps: a. Providing an acidic aqueous solution containing urine blue mother; and b. exposing the urine blue mother to a light source, wherein the wavelength of the light source is 300nm to 470nm, And wherein conversion of urocyanin to indigo is mediated solely by said light exposure.

2. The method according to claim 1, wherein: The uranin is exposed to sufficient radiant energy by the light source to convert at least 50% of the uranin to indigo.

3. A method for dyeing a product with indigo, comprising the following steps: a. Providing an acidic aqueous solution of urine blue mother; b. applying the urine blue mother solution in step (a) to the product; and c. exposing the product treated with urine blue masterbatch in step (b) to a light source to obtain a dyed product, Wherein, the wavelength of the light source is 300nm to 470nm, and Therein, the glycosidic bonds in the urocyanate are cleaved only by the light exposure.

4. The method according to any one of claims 1 to 3, wherein: The light source is selected from sunlight, artificial daylight bulbs, broadband light sources and LED lamps.

5. The method according to claim 3 or 4, wherein: The product treated with urine blue mother is illuminated by the light source at 20-220W / m 2 Irradiation.

6. The method according to any one of claims 3 to 5, wherein: The products treated with urea blue were exposed to the light source for up to 300 minutes.

7. The method according to any one of claims 3 to 6, wherein: The final radiation exposure of the product treated with urine blue masterbatch is 100-10000J / m 2 .

8. The method according to any one of claims 3 to 7, wherein: Light exposure occurs when the product is immersed or soaked in the urea blue solution.

9. The method according to any one of claims 3 to 8, wherein: The product is selected from one or more of yarn, textile and fabric, preferably a cotton-based product.

10. The method according to any one of claims 3 to 9, wherein: The product is a textile or fabric, and wherein the concentration of urea blue in the urea blue solution is 0.1-10 μmol / cm 2 textile or fabric.

11. The method according to claim 9 or 10, wherein: The product is immersed, soaked in, or coated with the urea blue master solution.

12. The method according to any one of claims 1 to 11, wherein: Step (a) comprises: Providing (i) indoxyl, (ii) nucleotide sugar and (iii) a polypeptide having glycosyltransferase activity; mixing components (i), (ii) and (iii), preferably under reaction conditions where less than 2% free oxygen is present; and The mixture is reacted to obtain urea blue mother.

13. A method for converting urea blue mother into indigo by light, wherein: The wavelength of the light is 300 nm to 450 nm, and wherein the urea blue is in an acidic aqueous solution.

14. The use of light according to claim 13, wherein: The indigo is simultaneously used to dye the product.

15. The use of light according to claim 14, wherein: The product is selected from one or more of yarn, textile and fabric.

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