Method and integrated system for the preparation of color pigments based on biocatalysis

Abstract

The application discloses a method and integrated system for preparing color pigments based on biological enzyme catalysis, and relates to the technical field of biological enzyme catalysis, and comprises providing a reaction system, wherein the reaction system comprises a neuroglobin mutant, an indole substrate and hydrogen peroxide, and the indole substrate is divided into a first substrate and a second substrate; the neuroglobin mutant is pre-reacted with the first part of the substrate and insufficient hydrogen peroxide to generate a pre-reaction solution containing an indole free radical intermediate, and then the second part of the substrate and the remaining hydrogen peroxide are added to perform a coupling reaction; the method and integrated system for preparing color pigments based on biological enzyme catalysis are characterized in that the reaction is divided into two steps, a part of the substrate is first converted into a free radical intermediate by using insufficient hydrogen peroxide, and then the substrate and hydrogen peroxide are added to complete directional coupling, so that the reaction path is adjusted from random oxidation to step-by-step controllable free radical coupling, the generation of by-products such as indole ketone and indigo is reduced, and the yield and chemical selectivity of indigo pigments are improved.

Description

Technical Field

[0001] This invention relates to the field of bio-enzyme catalysis technology, specifically to a method and integrated system for preparing colored pigments based on bio-enzyme catalysis. Background Technology

[0002] In recent years, the integration of biotechnology and chemical synthesis has opened up new pathways for the preparation of colored pigments. While traditional chemical synthesis methods can efficiently produce a variety of colored pigments, they are often accompanied by problems such as high energy consumption, high pollution, and difficulty in controlling product performance. In contrast, biocatalysis technology, with its mild reaction conditions, environmental friendliness, and high selectivity, has shown unique advantages in the field of organic synthesis, especially in the synthesis of chiral compounds and the preparation of pharmaceutical intermediates, where it has achieved remarkable results. Introducing biocatalysis technology into the preparation of colored pigments can not only achieve green synthesis, but also precisely control the structure and properties of pigment molecules through the specific catalytic action of enzymes, thereby developing novel colored pigments with excellent optical properties and stability.

[0003] However, in the practical application of bio-enzyme catalysis in the preparation of colored pigments, especially in the process of preparing indigo pigments by oxidative coupling of indole compounds using bio-enzymes, existing technologies face a key challenge: how to effectively control the reaction pathway to reduce the generation of byproducts. Because indole substrates are prone to random oxidation during the oxidation process, various non-target byproducts are generated, such as indoleone and indigo. The presence of these byproducts not only reduces the yield of the target indigo pigments but also seriously affects the color purity and chemoselectivity of the products. Therefore, improvements are needed. Summary of the Invention

[0004] The purpose of this invention is to provide a method and integrated system for preparing colored pigments based on bio-enzyme catalysis, in order to solve the problems in the prior art where the reaction path is difficult to control precisely when preparing indigo pigments by oxidative coupling of indole compounds using bio-enzyme catalysis, resulting in the formation of many by-products and low yield and color purity of the target pigment.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing colored pigments based on bio-enzyme catalysis, comprising the following steps:

[0006] S1. A reaction system is provided, the reaction system comprising a neuroglobin mutant with peroxidase activity, an indole substrate and hydrogen peroxide, wherein the indole substrate comprises a first substrate and a second substrate, the first substrate and the second substrate being the same or different indole compounds;

[0007] S2. The neuroglobin mutant is mixed with the first part of the substrate in a reaction medium, and the first part of hydrogen peroxide is added to carry out a pre-reaction to generate a pre-reaction solution containing an indole radical intermediate; wherein, the molar amount of the first part of hydrogen peroxide is less than the theoretical molar amount of hydrogen peroxide required to completely convert all the indole compounds in the first part of the substrate into indigo dimers.

[0008] S3. Add the remaining second portion of substrate and the second portion of hydrogen peroxide to the pre-reaction solution obtained in step S2, and continue the coupling reaction to generate indigo pigment products.

[0009] Furthermore, in step S2, the molar amount of the first portion of hydrogen peroxide is 50% to 90% of the theoretical molar amount required to convert all the indole molecules in the first portion of the substrate into indole radicals.

[0010] Furthermore, the molar ratio of the first portion of substrate to the second portion of substrate is 1:10 to 10:1.

[0011] Furthermore, the neuroglobin mutant is an A15C / H64D / F49Y triple mutant derived from human neuroglobin.

[0012] Furthermore, the first substrate and the second substrate are each independently selected from one of indole, 4-chloroindole, 5-chloroindole, 6-chloroindole, 7-chloroindole, 4-nitroindole, 5-nitroindole, and 6-bromoindole.

[0013] Furthermore, it also includes S4: during the coupling reaction in step S3, the Lab color value of the reaction solution is collected in real time using a color sensor. When the detected Lab color value reaches the preset target color range, the coupling reaction is terminated by stopping the addition of hydrogen peroxide and lowering the reaction temperature.

[0014] Furthermore, in step S4, when the detected Lab chromaticity value deviates from the preset target chromaticity range, a first substrate or a second substrate is added to the reaction system during step S3.

[0015] Furthermore, it also includes the following steps:

[0016] S5. A cellulose fiber fabric with indigo pigment particles attached after being subjected to the coupling reaction treatment in step S3.

[0017] S6. Immerse the fabric in a weakly acidic aqueous solution containing ascorbic acid, so that the indigo pigment particles adhering to the surface of the fabric are reduced in situ to water-soluble leuco pigments, which penetrate into the internal pores of the cellulose fibers.

[0018] S7. Remove the fabric from the solution and expose it to air or an oxygen-containing atmosphere to oxidize the leuco pigments that have penetrated into the fiber, regenerate indigo pigment crystals, and fix them inside the fiber.

[0019] Furthermore, in the weakly acidic aqueous solution of step S6, the concentration of ascorbic acid is from 0.5 g / L to 50 g / L, the pH value of the solution is from 3.0 to 6.5, and the treatment temperature is from 20°C to 60°C.

[0020] An integrated system for preparing colored pigments based on bio-enzyme catalysis includes:

[0021] A reaction vessel used to contain the reaction liquid;

[0022] The feeding unit is used to add the substrate and hydrogen peroxide to the reaction vessel in steps or continuously;

[0023] The detection unit includes a color sensor positioned facing the reaction solution for real-time acquisition of the Lab colorimetric value of the reaction solution;

[0024] The control unit is communicatively connected to the detection unit and the feeding unit, and is used to receive the Lab colorimetric value and send a feeding instruction to the feeding unit based on the comparison result of the Lab colorimetric value and the preset target colorimetric range.

[0025] The post-treatment unit is used to contain a weakly acidic aqueous solution containing ascorbic acid and to receive the fabric treated by the reaction vessel for immersion treatment in the weakly acidic aqueous solution.

[0026] Compared with existing technologies, the method and integrated system for preparing colored pigments based on bio-enzyme catalysis provided by this invention divides indole substrates into a first substrate and a second substrate. A neuroglobin mutant is first pre-reacted with the first substrate and an insufficient amount of hydrogen peroxide to generate a pre-reaction solution containing an indole radical intermediate. Then, the second substrate and the second hydrogen peroxide are added to this solution for a coupling reaction. This adjusts the reaction process from a random oxidation pathway to a stepwise, controllable radical coupling pathway, reducing the generation of monooxidation byproducts such as indoleone and indigo, and improving the yield and chemoselectivity of indigo pigments.

[0027] By using a color sensor to collect the Lab colorimetric value of the reaction solution in real time during the coupling reaction and comparing it with the preset target colorimetric range, the reaction is terminated by stopping the addition of hydrogen peroxide and lowering the reaction temperature when the colorimetric value reaches the target range. This transforms the reaction endpoint control from fixed time control to closed-loop control based on colorimetric parameters, reducing the color fluctuation of products between different batches.

[0028] By monitoring Lab color values ​​in real time during the coupling reaction, and adding a first or second substrate to the reaction system when the color value deviates from the preset target color range, the color trend of the reaction solution is dynamically corrected during the reaction process, so that the color of the final product can approach the preset target, improving the consistency of color between batches and the adjustability of intermediate hues.

[0029] After the coupling reaction is completed, the cellulose fiber fabric with indigo pigment particles is immersed in a weakly acidic aqueous solution containing ascorbic acid, so that the pigment particles are reduced in situ to water-soluble leuco and penetrate into the internal pores of the fiber. Then, the fabric is taken out and exposed to air, so that the leuco is re-oxidized into pigment crystals and fixed inside the fiber. This changes the form of the pigment on the fabric from physical surface adhesion to embedded crystals inside the fiber, thereby improving the rubbing fastness and washing fastness of the dyed fabric. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0031] Figure 1 A flowchart of the method provided in an embodiment of the present invention;

[0032] Figure 2 This is a system framework diagram provided for an embodiment of the present invention. Detailed Implementation

[0033] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0034] As attached Figure 1 To be continued Figure 2 As shown:

[0035] Example 1:

[0036] This invention provides a method for preparing colored pigments based on bio-enzyme catalysis. Experimental materials:

[0037] Enzyme protein:

[0038] The construction, expression, and purification of the neuroglobin triple mutant A15C / H64D / F49Y Ngb are as follows: Using the pET3a plasmid containing the wild-type human neuroglobin gene as a template, three site-directed mutations, A15C, H64D, and F49Y, were introduced sequentially using the Quikchange II site-directed mutagenesis kit. The forward primer for the F49Y mutation was 5'-AAC TGC CGC CAGTAC TCC AGC CCA GAG-3', and the reverse primer was 5'-CTC TGG GCT GGA GTA CTG GCG GCA GTT-3'. After verification of the mutation by DNA sequencing, the plasmid was transformed into *E. coli* BL21(DE3) competent cells and cultured in LB medium containing ampicillin at 37°C until the OD600 reached approximately 0.6–0.8. IPTG was then added to a final concentration of 0.5 mM, and expression was induced at 25°C for 12–16 hours. The bacterial cells were collected by centrifugation, resuspended in 100 mM potassium phosphate buffer (pH 7.0), and then treated with 1 mM PMSF and a small amount of DNase. After sonication, the cells were centrifuged at 4°C and 12000 rpm for 30 minutes, and the supernatant was collected. The supernatant was then purified sequentially using a DEAE-52 anion exchange column, a Sephacryl S-100 gel filtration column, and a Superdex 75 10 / 300 GL column. The fraction containing the target protein was collected. The purified protein was detected as a single band by SDS-PAGE, and the total protein molar extinction coefficient ε(406 nm) was determined to be 150 ± 3 mM using the hemochromatographic method. -1 ·cm -1 This is used for subsequent protein concentration determination.

[0039] Reagents:

[0040] Indole, purity ≥99%, dissolved in a small amount of anhydrous ethanol and diluted with deionized water to prepare a 100mM stock solution, to be used immediately; hydrogen peroxide, freshly diluted with deionized water to 100mM from a 30% stock solution, stored at 4℃ protected from light, to be used on the same day; potassium dihydrogen phosphate and dipotassium hydrogen phosphate, analytical grade, prepared as a 50mM potassium phosphate buffer solution, pH 7.0; dimethylformamide (DMF), chromatographic grade.

[0041] Main instruments:

[0042] The UV-Vis spectrophotometer is equipped with a constant temperature colorimetric rack; pH meter; constant temperature magnetic stirring water bath; high-speed centrifuge with a maximum speed of not less than 12000 r / min; and analytical balance with an accuracy of 0.1 mg.

[0043] Experimental methods:

[0044] Preparation of the reaction system:

[0045] Take a 10 mL round-bottom glass reaction flask and add 50 mM potassium phosphate buffer (pH 7.0) to approximately 80% of the final reaction volume (2 mL). Add A15C / H64D / F49Y Ngb enzyme solution to bring the final enzyme concentration to 10 μM. Place the reaction flask in a temperature-controlled magnetically stirred water bath at 37°C and stir gently.

[0046] Step-by-step feeding operation:

[0047] Step 1 (Pre-reaction stage):

[0048] Add the first portion of the substrate—indole mother liquor—to the reaction system to achieve a final indole concentration of 0.5 mM. After thorough mixing, add the first portion of hydrogen peroxide in a single injection using a microsyringe to achieve a final H₂O₂ concentration of 0.125 mM. The molar amount of this first portion of hydrogen peroxide is 50% of the theoretical molar amount required to convert all the indole in the first portion of the substrate into indole radicals. Start timing simultaneously with the addition of H₂O₂.

[0049] The reaction was carried out with gentle stirring at 37°C for 5 minutes. During this stage, the reaction solution gradually turned light yellow, indicating that indole was partially oxidized to form indole free radical intermediates, but no large amount of indigo precipitate had yet been formed. This process is the pre-reaction stage, and the resulting reaction solution is a pre-reaction solution containing indole free radical intermediates.

[0050] Step Two (Coupling Reaction Stage):

[0051] At the end of the pre-reaction phase (i.e., the end of the aforementioned 5 minutes), while maintaining the temperature and stirring, the second portion of the substrate—indole mother liquor—was added to the pre-reaction solution to bring the final concentration of indole in the second portion of the substrate to 0.5 mM (after combining the two portions of the substrate, the total amount of indole added to the system corresponds to a final concentration of 1.0 mM). Subsequently, the second portion of hydrogen peroxide was immediately added to bring the final concentration of H2O2 to 0.125 mM (the total amount of H2O2 added to the system corresponds to a final concentration of 0.25 mM).

[0052] Continue stirring gently at 37°C for 10 minutes. This stage is the coupling reaction stage, during which the reaction solution gradually changes from light yellow to blue-green, and finally a distinct blue granular precipitate appears, indicating the formation of indigo.

[0053] Separation and identification of reaction products:

[0054] Centrifugation of the product: After the reaction is complete, transfer all the reaction solution to a centrifuge tube and centrifuge at 10000 rpm for 10 minutes. After centrifugation, a blue precipitate will be visible at the bottom of the tube, and the supernatant will be pale yellow. Discard the supernatant (or retain it for further liquid chromatography analysis), and retain the blue precipitate.

[0055] Dissolution and Spectroscopic Identification of the Product: 2 mL of DMF was added to the above blue precipitate, and the mixture was shaken to dissolve. The precipitate quickly dissolved to form a transparent blue solution. This blue solution was then analyzed using a UV-Vis spectrophotometer in the wavelength range of 400–800 nm, with DMF as a blank reference. The obtained spectrum showed a characteristic absorption peak at 610 nm, which was consistent with the characteristic absorption peak of commercial indigo standards in DMF.

[0056] Meanwhile, a portion of the blue precipitate was analyzed by electrospray ionization mass spectrometry (ESI-MS) in negative ion mode, and the main signal peak was found to be m / z 261.18 [MH], similar to that of indigo (molecular formula C). 16 H 10 The [MH] peak of N2O2 (theoretical molecular weight 262.1 Da) matches, confirming that the product is indigo.

[0057] Yield calculation: The amount of unreacted indole residue in the supernatant after the reaction was determined by HPLC. The concentration of unreacted indole was found to be approximately 0.18 mM, therefore the total amount of indole consumed was approximately 0.82 mM.

[0058] Indigo yield is calculated using the following formula:

[0059] Indigo yield = (Indigo production × 2 / Total indole consumed) × 100%

[0060] The indigo formation amount was calculated by comparing the HPLC peak area after precipitation and dissolution with the indigo standard, and the indigo formation amount was approximately 0.34 mM. Substituting into the calculation, we get:

[0061] Indigo yield = (0.34 × 2 / 0.82) × 100% ≈ 82.9%.

[0062] In this embodiment, both the first and second substrates are indole, representing the case where the first and second substrates are the same compound. When the first and second substrates are different indole derivatives, the stepwise feeding sequence of this embodiment can be followed to prepare asymmetric coupling products or mixed-color pigments. The proportion of hydrogen peroxide in the first part is not limited to 50%; adjusting it within the range of 50% to 90% can effectively complete the pre-reaction stage. The specific proportion can be flexibly set according to the selectivity requirements of the target product.

[0063] Example 2:

[0064] This embodiment illustrates the specific implementation process of the color feedback control method in the catalytic synthesis of 5,5'-dichloroindole from 5-chloroindole, including the following experimental materials:

[0065] Enzyme protein:

[0066] The neuroglobin triple mutant A15C / H64D / F49Y Ngb was constructed, expressed, and purified using the same methods as in Example 1.

[0067] Reagents:

[0068] 5-Chloroindole, purity ≥98%, dissolved in a small amount of anhydrous ethanol and diluted with deionized water to prepare a 100mM stock solution, to be used immediately; Hydrogen peroxide, freshly diluted with deionized water to 100mM from a 30% stock solution, stored at 4℃ protected from light, to be used on the same day; Potassium dihydrogen phosphate and dipotassium hydrogen phosphate, analytical grade, prepared as a 50mM potassium phosphate buffer solution, pH 7.0; Dimethylformamide (DMF), chromatographic grade.

[0069] Main instruments:

[0070] The system includes: a UV-Vis spectrophotometer; a pH meter; a thermostatically heated magnetically stirred water bath; a high-speed centrifuge; an analytical balance; a reflective color sensor capable of detecting and outputting CIE Lab* color values ​​in a wavelength range of 400–700 nm, using a D65 standard light source with a 10° observation angle, and the sensor probe positioned 15 mm horizontally from the center of the reaction liquid surface; a microcontroller control module connected to the color sensor via a serial communication interface, with a pre-set color comparison program; two micro-injection pumps, one for replenishing the substrate solution and the other for replenishing the hydrogen peroxide solution, both electrically connected to the microcontroller control module and capable of accepting start / stop commands; and a cylindrical transparent glass bottle with an outer diameter of 25 mm, a wall thickness of 1.2 mm, and a volume of 10 mL.

[0071] Experimental methods:

[0072] Reaction system preparation and equipment commissioning:

[0073] Take the reaction vessel and add 50mM potassium phosphate buffer (pH 7.0) to approximately 80% of the final reaction volume (2mL). Add A15C / H64D / F49Y Ngb enzyme solution to bring the final enzyme concentration to 10μM. Place the reaction vessel in a temperature-controlled magnetically stirred water bath at 37°C and stir gently.

[0074] Mount the reflective color sensor onto the bracket, aligning the sensor probe with the outer wall of the reaction vessel, with the probe tip approximately 5mm from the outer wall. Align the center of the sensor window horizontally with the middle of the reaction liquid surface. Allow the sensor to warm up for 10 minutes, then perform a blank calibration using an empty reaction vessel filled with an equal volume of buffer solution.

[0075] The outlet lines of micro-injection pump 1 (substrate pump) containing 5-chloroindole mother liquor and micro-injection pump 2 (oxidant pump) containing 100mM hydrogen peroxide solution are fixed above the reaction vessel opening, with the outlets extending about 10mm above the liquid surface to ensure that the added droplets fall directly into the reaction solution.

[0076] The microcontroller control module is pre-programmed with the target color range: L value [15,30], a value [0,8], b* value [-35,-20]. This range corresponds to the typical colorimetric characteristics of 5,5'-dichloroindigo in the reaction solution.

[0077] Stepwise feeding pre-reaction:

[0078] The first portion of the substrate—5-chloroindole stock solution—was added to the reaction system to a final concentration of 0.5 mM. After thorough stirring, the first portion of hydrogen peroxide was manually added in a single injection using a microsyringe to bring the final H₂O₂ concentration to 0.125 mM. Timing was started simultaneously with the addition of H₂O₂, and the reaction was carried out with gentle stirring at 37°C for 5 minutes. During this stage, the reaction solution gradually turned a pale purple-red color, indicating that 5-chloroindole was partially oxidized to generate the corresponding free radical intermediate. This process is the pre-reaction stage.

[0079] Coupling reaction and chromaticity feedback control:

[0080] At the end of the pre-reaction phase (i.e., the end of the aforementioned 5 minutes), manually add the second portion of substrate—5-chloroindole stock solution—to the pre-reaction solution in one go, bringing the final concentration to 0.5 mM (after combining the two substrate portions, the total amount of 5-chloroindole added to the system corresponds to a final concentration of 1.0 mM). Simultaneously, manually add the second portion of hydrogen peroxide in one go, bringing the final H2O2 concentration to 0.125 mM. Continue to maintain gentle stirring at 37°C; the coupling reaction will then begin.

[0081] From the start of the coupling reaction, an automatic monitoring program is initiated by the color sensor and the microcontroller control module. The color sensor collects the Lab* chromaticity value of the reaction solution every 5 seconds and sends the data to the microcontroller control module in real time. The microcontroller control module compares each received chromaticity value with a preset target chromaticity range.

[0082] First assessment (60 seconds after the start of the reaction): Sensor values ​​L*=48, a*=2, b*=-8, not within the target range. The microcontroller did not issue an intervention command, and the reaction continued.

[0083] Second assessment (120 seconds after the start of the reaction): Sensor detection values ​​L*=42, a*=3, b*=-14. The b* value is trending towards the lower limit of the target range but has not yet entered it. The microcontroller did not issue an intervention command.

[0084] The third assessment (180 seconds after the reaction started): Sensor values ​​L*=32, a*=4, b*=-22. At this point, all three parameters are within the preset target color range (L* value 32 falls within the adjacent interval of [15,30], a value 4 falls within [0,8], and b value -22 falls within [-35,-20]). The microcontroller control module immediately issues two commands: First, it sends a stop command to the oxidant pump to stop the addition of hydrogen peroxide; second, it triggers the cooling solenoid valve to allow low-temperature cooling water (approximately 2°C) to flow into the outer jacket of the reaction vessel, lowering the reaction solution temperature to below 5°C within approximately 30 seconds, inhibiting enzyme activity, and terminating the coupling reaction.

[0085] After the reaction was terminated, the final color values ​​were recorded: L*=28, a*=4, b*=-23.

[0086] Verification of feed intervention under deviation conditions:

[0087] To verify the effectiveness of the feed-off function in claim 7, another set of experiments was conducted using the same equipment and under the same initial reaction conditions. In this set of experiments, at 180 seconds into the coupling reaction, the sensor values ​​L*=45, a*=1, and b*=-10, significantly deviating from the preset target color range. The microcontroller control module calculated that the b* value was higher than the upper limit of the target range, requiring substrate replenishment to push the reaction towards the blue direction. The control module sent a command to the substrate pump to inject 25 μL of 5-chloroindole stock solution in a single pulse (increasing the substrate concentration in the reaction system by approximately 0.05 mM). Monitoring continued, and at 300 seconds, the values ​​L*=30, a*=5, and b*=-25, with all three parameters falling into the target range. The control module then terminated the reaction as described above.

[0088] Product Confirmation: After the reaction was terminated, the reaction solution was transferred to a centrifuge tube and centrifuged at 10000 r / min for 10 minutes. A purple precipitate was visible at the bottom of the tube after centrifugation. The supernatant was discarded, and 2 mL of DMF was added to the precipitate. The solution was shaken to dissolve the precipitate and form a transparent purple solution. Using DMF as a blank reference, a UV-Vis spectrophotometer was used to scan the spectrum in the wavelength range of 400–800 nm. The obtained spectrum showed a characteristic absorption peak at 560 nm, which was consistent with the absorption peak reported in the literature for 5,5'-dichloroindigo. The filtrate was analyzed by ESI-MS in negative ion mode. The main signal peak was measured to be m / z 329.1 [MH], which is consistent with the absorption peak of 5,5'-dichloroindigo (molecular formula C 16 The [MH] peak of H8Cl2N2O2 (theoretical molecular weight 330 Da) matches, confirming that the product is the target pigment.

[0089] In this embodiment, the preset target color range is set for a single product, 5,5'-dichloroindigo. When using different indole derivatives as substrates, different target color ranges can be pre-defined based on the characteristic color of the corresponding products and then written into the microcontroller control module for application. In addition to supplementing the substrate, the feeding intervention strategy can also determine whether hydrogen peroxide needs to be supplemented based on the actual color deviation direction. The criterion program can be flexibly programmed according to the reaction kinetic curves of different substrates.

[0090] Example 3:

[0091] This embodiment illustrates the specific implementation process of acid post-treatment to reduce indigo leuco in situ and crystallize within the fiber to improve fastness, including the following experimental materials:

[0092] Enzyme protein:

[0093] The neuroglobin triple mutant A15C / H64D / F49Y Ngb was constructed, expressed, and purified using the same methods as in Example 1.

[0094] Reagents:

[0095] Indole, purity ≥99%, dissolved in a small amount of anhydrous ethanol and diluted with deionized water to prepare a 100mM stock solution, to be used immediately; Hydrogen peroxide, freshly diluted with deionized water to 100mM from a 30% stock solution, stored at 4℃ protected from light, to be used on the same day; Potassium dihydrogen phosphate and dipotassium hydrogen phosphate, analytical grade, prepared as a 50mM potassium phosphate buffer solution, pH 7.0; L-ascorbic acid, food grade, purity ≥99%; Citric acid, analytical grade, used to adjust the pH of the post-treatment solution; Deionized water.

[0096] Fabric material:

[0097] Pure white cotton fabric, plain weave, approximately 150g / m², treated with desizing, scouring, and bleaching; contains no fluorescent whitening agents. Cut into 5cm x 5cm square samples for later use.

[0098] Main instruments:

[0099] UV-Vis spectrophotometer; pH meter; constant temperature magnetic stirring water bath; high-speed centrifuge; analytical balance; electric heating drying oven; constant temperature shaking water bath with temperature control accuracy ±1℃; friction fastness tester with a friction head diameter of 16mm, applied force of 9N, and reciprocating stroke of 100mm.

[0100] Experimental methods:

[0101] Preparation of indigo-dyed fabrics:

[0102] Take a 50mL round-bottom glass reaction flask and add 50mM potassium phosphate buffer (pH 7.0) to approximately 80% of the final reaction volume (20mL). Add A15C / H64D / F49Y Ngb enzyme solution to bring the final enzyme concentration to 10μM. Add indole stock solution to the reaction system to bring the final indole concentration to 2mM. Completely immerse a 5cm × 5cm piece of pure white cotton fabric in the reaction solution. Place the reaction flask in a constant-temperature magnetically stirred water bath at 37℃ and stir gently. Add hydrogen peroxide in a single injection using a microsyringe to bring the final H2O2 concentration to 1mM. Continue the reaction at 37℃ with stirring for 60 minutes. During the reaction, the reaction solution gradually turns blue, and the surface of the cotton fabric is simultaneously colored. After the reaction is complete, remove the cotton fabric, gently rinse twice with deionized water to remove loosely attached pigment particles, and allow it to air dry naturally. The resulting fabric is a fabric to be treated that has been initially dyed with indigo. Its surface is covered with indigo pigment particles and is blue in color, but there is some surface floating color. A small amount of blue can be seen to come off when lightly rubbed with a finger.

[0103] Preparation of ascorbic acid weakly acidic post-treatment solution:

[0104] Weigh 10.0 g of L-ascorbic acid and dissolve it in approximately 800 mL of deionized water, stirring until completely dissolved. Adjust the pH of the solution to 5.0 with citric acid, then bring the volume to 1000 mL with deionized water. The resulting post-treatment solution has an ascorbic acid concentration of 10 g / L and a pH of 5.0. Transfer the post-treatment solution to a covered glass or stainless steel container and preheat it to 40°C in a constant-temperature shaking water bath.

[0105] Fabric post-processing operations:

[0106] After the temperature of the post-treatment solution stabilizes at 40℃, the fabric to be treated, which has undergone initial indigo dyeing, obtained from the preparation step of the indigo-dyed fabric, is completely immersed in the post-treatment solution. The ratio of fabric to post-treatment solution is 1:50 (i.e., 50 mL of post-treatment solution per gram of fabric). The container is covered and treated at 40℃ with shaking at a rate of 60 times per minute for 15 minutes.

[0107] Approximately 3 minutes after the treatment began, the fabric color started to lighten. By 10 minutes, the fabric color had noticeably faded to a light bluish-gray. By 15 minutes, the fabric color had become extremely pale, almost white. This color change indicates that the indigo particles adhering to the fabric surface and superficial layers were reduced in situ by ascorbic acid to water-soluble leuco forms under weakly acidic conditions. These leuco forms dissolved from the fabric surface and penetrated into the micropores, cavities, and amorphous regions within the cotton fibers according to the concentration gradient.

[0108] After treatment, the fabric is removed from the post-treatment solution using tweezers, excess liquid is drained, and the fabric is laid flat on a stainless steel wire mesh and placed in a fume hood for natural oxidation. After approximately 5 minutes of exposure to air, the fabric begins to slowly regain its blue color; after approximately 20 minutes, the blue color is restored to a depth similar to that before treatment; after approximately 30 minutes, the color is fully restored and is more uniform and deeper than before treatment. The oxidized fabric is then gently rinsed once with deionized water to remove residual ascorbic acid and placed in the air to dry naturally again. The post-treatment solution can be recycled for the next batch of fabric, and its reducing power can be regenerated by supplementing with an appropriate amount of ascorbic acid.

[0109] Fabric fastness test:

[0110] Fabrics that have undergone complete post-treatment as described above and untreated initially dyed fabrics (i.e., directly dried fabrics obtained from the preparation steps of indigo-dyed fabrics) were subjected to dry rubbing fastness tests according to the methods specified in GB / T 3920-2008 "Textiles - Tests for Color Fastness - Color Fastness to Rubbing". The standard cotton lining fabric for rubbing was 5cm × 5cm in size.

[0111] Test conditions: The friction head applies a downward pressure of 9N and rubs the sample back and forth 10 times along its length at a rate of 1 reciprocating cycle per second, with a reciprocating stroke of 100mm. After the test, the staining grade of the standard cotton lining fabric for rubbing is evaluated using the gray scale for staining assessment (GB / T 251).

[0112] Test results showed that the initial dyed fabric without post-treatment had a dry rubbing staining level of 3, with obvious blue staining on the lining fabric. The fabric post-treated using the post-treatment steps in this embodiment had a dry rubbing staining level of 4, with only slight blue traces on the lining fabric.

[0113] Two more samples of the two fabrics were taken, cut into small pieces, and boiled in equal amounts of boiling water for 10 minutes. The color fading was then observed. In the untreated fabric, the blue dye clearly dissolved during boiling, and the boiling solution was pale blue. In contrast, the treated fabric produced a virtually colorless and transparent boiling solution. These observations indicate that the ascorbic acid weak acid post-treatment transforms the indigo pigment from a physical adhesion to the fiber surface to an embedded fixation within the fiber, thus effectively improving color fastness.

[0114] In this embodiment, the ascorbic acid concentration is 10 g / L, which is within the range of 0.5 g / L to 50 g / L in this invention. For fabrics with darker colors and higher indigo adhesion, the ascorbic acid concentration can be appropriately increased or the treatment time extended. The pH value of the post-treatment solution is 5.0, which is within the range of 3.0 to 6.5 in this invention. Too low a pH value may lead to a decrease in cotton fiber strength, while too high a pH value will slow down the reduction rate. The pH value can be adjusted within this range according to actual needs during operation. The treatment temperature is 40℃, which is within the range of 20℃ to 60℃ in this invention. Increasing the temperature can accelerate the reduction and penetration rate, but it should not exceed 60℃ to avoid excessive decomposition of ascorbic acid. The post-treatment solution can be recycled. When the reduction efficiency decreases, adding an appropriate amount of ascorbic acid to the solution and readjusting the pH value can restore its function. This embodiment focuses on indigo synthesized using indole as a substrate. However, this post-processing method is also applicable to indigo pigments synthesized from other indole derivatives (such as 4,4'-dichloroindigo, 5,5'-dichloroindigo, 6,6'-dibromoindigo, etc.).

[0115] Example 4:

[0116] This embodiment illustrates the specific implementation process of preparing multicolor indigo pigments using a stepwise feeding process when different substituted indole derivatives are used as substrates, including the following experimental materials:

[0117] Enzyme protein:

[0118] The neuroglobin triple mutant A15C / H64D / F49Y Ngb was constructed, expressed, and purified using the same methods as in Example 1.

[0119] Reagents:

[0120] 4-Chloroindole, purity ≥98%, dissolved in a small amount of anhydrous ethanol and diluted with deionized water to prepare a 100mM stock solution, to be used immediately; 4-Nitroindole, purity ≥98%, dissolved in a small amount of anhydrous ethanol and diluted with deionized water to prepare a 100mM stock solution, to be used immediately; 6-Bromoindole, purity ≥98%, dissolved in a small amount of anhydrous ethanol and diluted with deionized water to prepare a 100mM stock solution, to be used immediately; Hydrogen peroxide, freshly diluted with deionized water to 100mM from a 30% stock solution, stored at 4℃ protected from light, to be used on the same day; Potassium dihydrogen phosphate and dipotassium hydrogen phosphate, analytical grade, prepared as a 50mM potassium phosphate buffer solution, pH 7.0; Dimethylformamide, chromatographic grade.

[0121] Main instruments:

[0122] The instrument is equipped with a UV-Vis spectrophotometer with a constant temperature colorimetric rack, a pH meter, a constant temperature magnetic stirring water bath, a high-speed centrifuge with a maximum speed of not less than 12000 r / min, an analytical balance with an accuracy of 0.1 mg, and a high-resolution mass spectrometer with an electrospray ionization source and negative ion mode detection.

[0123] Experimental methods:

[0124] Synthesis of 4,4'-dichloroindole as a substrate:

[0125] Take a 10 mL round-bottom glass reaction flask and add 50 mM potassium phosphate buffer (pH 7.0) to approximately 80% of the final reaction volume (2 mL). Add A15C / H64D / F49Y Ngb enzyme solution to bring the final enzyme concentration to 10 μM. Place the reaction flask in a temperature-controlled magnetically stirred water bath at 37°C and stir gently.

[0126] The first substrate, 4-chloroindole stock solution, was added to the reaction system to bring the final concentration of 4-chloroindole in the first substrate to 0.5 mM. After stirring thoroughly, the first portion of hydrogen peroxide was added in a single injection using a microsyringe to bring the final concentration of H2O2 to 0.125 mM. The reaction was carried out with gentle stirring at 37°C for 5 minutes. During this stage, the reaction solution gradually turned light blue-gray, indicating that 4-chloroindole was partially oxidized to generate the corresponding free radical intermediate.

[0127] After the pre-reaction phase timed out, the second substrate—4-chloroindole mother liquor—was added to the pre-reaction solution to bring the final concentration of 4-chloroindole in the second substrate to 0.5 mM (after combining the two substrates, the total amount of 4-chloroindole added to the system corresponds to a final concentration of 1.0 mM). Then, the second portion of hydrogen peroxide was immediately added to bring the final concentration of H₂O₂ to 0.125 mM. The mixture was then stirred gently at 37°C for 10 minutes. The reaction solution gradually changed from light blue-gray to bright blue, and a blue granular precipitate appeared.

[0128] After the reaction was complete, all the reaction solution was transferred to centrifuge tubes and centrifuged at 10000 r / min for 10 minutes. A bright blue precipitate was visible at the bottom of the tube after centrifugation. The supernatant was discarded, and the precipitate was retained. 2 mL of DMF was added to the precipitate, and the mixture was shaken to dissolve, forming a clear, bright blue solution. Using DMF as a blank reference, a UV-Vis spectrophotometer was used to scan the spectrum in the wavelength range of 400–800 nm. The obtained spectrum showed a characteristic absorption peak at 630 nm. The filtrate was analyzed by ESI-MS in negative ion mode. The main signal peak was measured to be m / z 329.1 [MH], which is related to 4,4'-dichloroindigo (molecular formula C...). 16 H8C l2 The [MH] peak of N2O2 (theoretical molecular weight 330 Da) matches.

[0129] Synthesis of 4,4'-dinitroindigo from 4-nitroindole:

[0130] Take another 10 mL round-bottom glass reaction flask and proceed with the same steps as the synthesis of 4,4'-dichloroindole using 4-chloroindole as the substrate, but replace the substrate with 4-nitroindole. The final concentration of 4-nitroindole in the first part of the substrate is 0.5 mM. Add the first part of H₂O₂ to a final concentration of 0.125 mM, and react at 37°C for 5 minutes. The reaction solution gradually turns light yellowish-brown. After the pre-reaction is complete, add the second part of 4-nitroindole to a final concentration of 0.5 mM, and add the second part of H₂O₂ to a final concentration of 0.125 mM. Continue the reaction for 10 minutes. The reaction solution changes from light yellowish-brown to dark yellow, and a yellow granular precipitate appears.

[0131] After the reaction, centrifugation yielded a yellow precipitate. The precipitate dissolved in 2 mL of DMF to form a transparent yellow solution, and a characteristic absorption peak was observed at 580 nm in the spectral scan. ESI-MS analysis in negative ion mode revealed a major signal peak at m / z 351.2 [MH], similar to 4,4'-dinitroindigo (molecular formula C...). 16 The [MH] peak of H6N4O6 (theoretical molecular weight 352 Da) matches.

[0132] Synthesis of 6,6'-dibromoindigo from 6-bromoindole:

[0133] Take a third 10mL round-bottom glass reaction flask and proceed with the same steps as the synthesis of 4,4'-dichloroindole using 4-chloroindole as the substrate, but replace the substrate with 6-bromoindole. The final concentration of 6-bromoindole in the first part of the substrate is 0.5mM. Add the first part of H₂O₂ to a final concentration of 0.125mM, and react at 37°C for 5 minutes. The reaction solution gradually turns light purple-gray. After the pre-reaction is complete, add the second part of 6-bromoindole to a final concentration of 0.5mM, and add the second part of H₂O₂ to a final concentration of 0.125mM. Continue the reaction for 10 minutes. The reaction solution changes from light purple-gray to purple-red, and a purple granular precipitate appears.

[0134] After the reaction, centrifugation yielded a purple precipitate. The precipitate dissolved in 2 mL of DMF to form a transparent purple solution, and a characteristic absorption peak was observed at 535 nm in the spectral scan. ESI-MS analysis in negative ion mode revealed a major signal peak at m / z 419.2 [MH], consistent with 6,6'-dibromoindigo (molecular formula C...). 16 The [MH] peak of H8Br2N2O2 (theoretical molecular weight 420 Da) matches.

[0135] Synthesis of mixed-color pigments using 4-chloroindole and 4-nitroindole as common substrates:

[0136] Take the fourth 10mL round-bottom glass reaction flask and prepare the enzyme buffer using the same initial steps as for the synthesis of 4,4'-dichloroindigo using 4-chloroindole as the substrate. In this experiment, the first part of the substrate consists of 4-chloroindole and 4-nitroindole in a molar ratio of 2:1, with a final concentration of 0.33mM for 4-chloroindole and 0.17mM for 4-nitroindole. The total concentration of indole derivatives in the first part of the substrate is 0.5mM. Add the first part of H2O2 to 0.125mM and react at 37℃ for 5 minutes. The reaction solution exhibits an intermediate hue between blue-gray and yellowish-brown.

[0137] After the pre-reaction was completed, the second substrate was added, with the same composition and ratio as the first substrate: a molar ratio of 4-chloroindole to 4-nitroindole of 2:1. After the addition, the final total concentration of 4-chloroindole in the combined system was 0.67 mM, and the final total concentration of 4-nitroindole was 0.33 mM. The second portion of H₂O₂ was added to a final concentration of 0.125 mM, and the reaction was continued for 10 minutes. The reaction solution gradually turned green, and a green granular precipitate appeared.

[0138] Centrifugation after the reaction yielded a green precipitate. The precipitate dissolved in 2 mL of DMF to form a transparent green solution. Spectroscopic scanning revealed a broad peak between 610 nm and 580 nm, exhibiting both blue and yellow absorption characteristics. ESI-MS analysis in negative ion mode revealed two main signal peaks at m / z 329.1 and m / z 351.2, corresponding to the [MH] peaks of 4,4'-dichloroindigo and 4,4'-dinitroindigo. No signal peak for the heterosubstituted product of 4-chloro-4'-nitroindigo was detected near m / z 340, indicating that the coupling reaction tends to occur between molecules of the same substrate, and the product is a mixture of two indigo derivatives, whose co-existence gives the solution and precipitate their green color.

[0139] Summary of product color characteristics:

[0140] The products obtained from the above four reactions were dissolved in equal volumes of DMF, and the colors were observed visually. The results are as follows: the 4,4'-dichloroindole solution obtained using 4-chloroindole as a substrate was bright blue; the 4,4'-dinitroindole solution obtained using 4-nitroindole as a substrate was yellow; the 6,6'-dibromoindole solution obtained using 6-bromoindole as a substrate was purple; and the mixed product solution obtained using a 2:1 mixture of 4-chloroindole and 4-nitroindole as substrates was green. These four colors correspond to the blue, yellow, purple, and green color families, respectively. Combined with the blue indole product obtained using indole as a substrate in Example 1, the preparation of multi-colored indole pigments was achieved.

[0141] In this embodiment, the stepwise addition of each substituted indole substrate uses the same reaction parameters as for indole. Specifically, the amount of hydrogen peroxide in the first portion is 50% of the theoretical amount required to convert all the indole derivatives in the first portion of the substrate into free radicals; the pre-reaction time is 5 minutes; and the coupling reaction time is 10 minutes. For indole derivatives with different substituents, the pre-reaction time and hydrogen peroxide ratio can be adjusted appropriately based on these parameters according to their reactivity. The types of substrates listed in this embodiment are not exhaustive. Other indole derivatives of claim 5 (such as 5-chloroindole, 6-chloroindole, 7-chloroindole, 5-nitroindole, etc.) can all be coupled and oxidized using the same stepwise addition method to obtain indigo pigments of the corresponding colors. The ratio of the common substrate is not limited to 2:1. By changing the molar ratio of the two substrates, the color tendency of the final mixed product can be adjusted within a certain range.

[0142] Comparative Example 1:

[0143] This comparative example is used to illustrate the process of synthesizing indigo from indole using A15C / H64D / F49Y Ngb catalysis via a one-time feeding method, and serves as a reference to the stepwise feeding process in Example 1.

[0144] Experimental materials:

[0145] Enzyme protein:

[0146] The neuroglobin triple mutant A15C / H64D / F49Y Ngb was constructed, expressed, and purified using the same methods as in Example 1.

[0147] Reagents:

[0148] Indole, purity ≥99%, dissolved in a small amount of anhydrous ethanol and diluted with deionized water to prepare a 100mM stock solution, to be used immediately; hydrogen peroxide, freshly diluted with deionized water to 100mM from a 30% stock solution, stored at 4℃ protected from light, to be used on the same day; potassium dihydrogen phosphate and dipotassium hydrogen phosphate, analytical grade, prepared as a 50mM potassium phosphate buffer solution, pH 7.0; dimethylformamide (DMF), chromatographic grade.

[0149] Main instruments:

[0150] The UV-Vis spectrophotometer is equipped with a constant temperature colorimetric rack; pH meter; constant temperature magnetic stirring water bath; high-speed centrifuge with a maximum speed of not less than 12000 r / min; and analytical balance with an accuracy of 0.1 mg.

[0151] Experimental methods:

[0152] Preparation of the reaction system:

[0153] Take a 10 mL round-bottom glass reaction flask and add 50 mM potassium phosphate buffer (pH 7.0) to approximately 80% of the final reaction volume (2 mL). Add A15C / H64D / F49Y Ngb enzyme solution to bring the final enzyme concentration to 10 μM. Place the reaction flask in a temperature-controlled magnetically stirred water bath at 37°C and stir gently.

[0154] One-time feeding operation:

[0155] Add the indole stock solution to the reaction system all at once to bring the final indole concentration to 1.0 mM. After stirring thoroughly, add all the hydrogen peroxide at once using a microsyringe to bring the final H2O2 concentration to 0.25 mM. Start timing simultaneously with the addition of H2O2.

[0156] The reaction was carried out with gentle stirring at 37°C for 15 minutes. Approximately 2 minutes after the start of the reaction, the reaction solution changed from colorless to pale yellow; after about 5 minutes, a blue-green turbidity appeared; and after about 10 minutes, a distinct blue granular precipitate was visible. The reaction was stopped after 15 minutes.

[0157] Separation and identification of reaction products:

[0158] Centrifugal separation of products:

[0159] After the reaction is complete, transfer all the reaction solution to centrifuge tubes and centrifuge at 10000 rpm for 10 minutes. A blue precipitate will be visible at the bottom of the tube after centrifugation, and the supernatant will be light yellow to light brown. Discard the supernatant and retain the blue precipitate.

[0160] Dissolution and spectroscopic identification of the product:

[0161] Add 2 mL of DMF to the above blue precipitate, shake to dissolve, and the precipitate dissolves to form a blue solution. Using DMF as a blank reference, perform a spectral scan in the wavelength range of 400 to 800 nm using a UV-Vis spectrophotometer. The obtained spectrum shows a characteristic absorption peak at a wavelength of 610 nm, which is consistent with the characteristic absorption peak of the indigo standard. ESI-MS analysis in negative ion mode revealed a major signal peak of m / z 261.18 [MH], confirming that the product is indigo.

[0162] Yield and byproduct analysis:

[0163] The residual amount of unreacted indole and the content of byproducts in the supernatant after the reaction were determined by HPLC. HPLC conditions were as described in the relevant section of the instruction manual. The concentration of unreacted indole was approximately 0.17 mM, and the total amount of indole consumed was approximately 0.83 mM. The amount of indigo formed was approximately 0.28 mM.

[0164] Indigo yield is calculated using the following formula: Indigo yield = (Indigo produced × 2 / Total indole consumed) × 100%. Substituting this into the formula, we get indigo yield = (0.28 × 2 / 0.83) × 100% ≈ 67.5%.

[0165] The supernatant was found to contain approximately 15 μM of indoleone, approximately 10 μM of indigo, and trace amounts of other oxidation byproducts.

[0166] Under single-feed conditions, the indigo yield was approximately 67.5%, and byproducts such as indoleone and indigo were detected in the supernatant of the reaction solution. This result is compared with the approximately 82.9% yield obtained in Example 1 with stepwise feeding; the yield was lower and the byproduct content was relatively higher with single-feed conditions.

[0167] Comparative Example 2:

[0168] This comparative example illustrates the process of synthesizing 5,5'-dichloroindole using A15C / H64D / F49Y Ngb catalysis without external chromaticity feedback control, and the batch-to-batch color consistency, serving as a reference to the chromaticity feedback control method in Example 2.

[0169] Experimental materials:

[0170] Enzyme protein:

[0171] The neuroglobin triple mutant A15C / H64D / F49Y Ngb was constructed, expressed, and purified using the same methods as in Example 1.

[0172] Reagents:

[0173] 5-Chloroindole, purity ≥98%, dissolved in a small amount of anhydrous ethanol and diluted with deionized water to prepare a 100mM stock solution, to be used immediately; hydrogen peroxide, freshly diluted with deionized water to 100mM from a 30% stock solution, stored at 4℃ protected from light, to be used on the same day; potassium dihydrogen phosphate and dipotassium hydrogen phosphate, analytical grade, prepared as a 50mM potassium phosphate buffer solution, pH 7.0; dimethylformamide, chromatographic grade.

[0174] Main instruments:

[0175] UV-Vis spectrophotometer; pH meter; thermostatic magnetic stirring water bath; high-speed centrifuge; analytical balance; reflective color sensor for endpoint colorimetric determination, not involved in process control.

[0176] Experimental methods:

[0177] Preparation and operation of the reaction system:

[0178] Take the reaction vessel and add 50mM potassium phosphate buffer (pH 7.0) to approximately 80% of the final reaction volume (2mL). Add A15C / H64D / F49Y Ngb enzyme solution to bring the final enzyme concentration to 10μM. Place the reaction vessel in a temperature-controlled magnetically stirred water bath at 37°C and stir gently.

[0179] Add 5-chloroindole stock solution to the reaction system in one go to achieve a final concentration of 1.0 mM. After stirring thoroughly, add hydrogen peroxide in one go using a microsyringe to achieve a final H2O2 concentration of 0.5 mM. Start timing simultaneously with the addition of H2O2 and continue stirring gently at 37°C for 15 minutes.

[0180] Repeat the operation three times under the same conditions, and label them batch A, batch B, and batch C respectively.

[0181] Reaction endpoint determination:

[0182] After 15 minutes of reaction, stop stirring and transfer all the reaction solution to centrifuge tubes. Centrifuge at 10000 rpm for 10 minutes. Discard the supernatant after centrifugation and retain the precipitate. Add 2 mL of DMF to each batch of precipitate and shake to dissolve. Measure the Lab* colorimetric values ​​of the three batches of DMF solutions using a color sensor and record the results as follows:

[0183] Batch A: L*=27, a*=6, b*=-22;

[0184] Batch B: L*=35, a*=2, b*=-16;

[0185] Batch C: L*=30, a*=4, b*=-24.

[0186] Product identification:

[0187] A batch of product A was subjected to UV-Vis spectroscopy, and a characteristic absorption peak was observed at 560 nm. ESI-MS analysis revealed that the main signal peak was 329.1 m / z [MH], which is similar to 5,5'-dichloroindigo (molecular formula C). 16 The [MH] peak of H8Cl2N2O2 matches, confirming the correct structure of the product.

[0188] Results Explanation:

[0189] Under conditions of a fixed reaction time of 15 minutes and no in-process intervention, the Lab values ​​of the three batches of product DMF solutions showed significant differences. Batch A had a color close to that of the product obtained through feedback control in Example 2, while batch B had a b value of -16, indicating a significant lack of blue hue, and batch C had a slightly higher L* value. The large color fluctuations among the three batches suggest that controlling the reaction endpoint with a fixed time alone is insufficient to guarantee color consistency across different batches.

[0190] Comparative Example 3:

[0191] This comparative example is used to illustrate the performance of indigo-dyed fabrics without acid post-treatment in terms of color fastness, and serves as a reference to the ascorbic acid weak acid post-treatment method in Example 3.

[0192] Experimental materials:

[0193] Fabric material:

[0194] Pure white cotton fabric, with the same specifications and treatment as in Example 3, is cut into 5cm×5cm square samples.

[0195] Reagents:

[0196] Indole, purity ≥99%; hydrogen peroxide, 30% stock solution freshly diluted to 100mM; potassium dihydrogen phosphate and dipotassium hydrogen phosphate, analytical grade, prepared as 50mM potassium phosphate buffer, pH 7.0; A15C / H64D / F49Y Ngb enzyme solution, prepared in the same way as in Example 1.

[0197] Main instruments:

[0198] UV-Vis spectrophotometer; pH meter; constant temperature magnetic stirring water bath; electric heating drying oven; friction fastness tester, parameters are the same as in Example 3.

[0199] Experimental methods:

[0200] Preparation of indigo-dyed fabrics:

[0201] Take a 50 mL round-bottom glass reaction flask and add 50 mM potassium phosphate buffer (pH 7.0) until the final reaction volume is approximately 80% of 20 mL. Add A15C / H64D / F49Y Ngb enzyme solution to bring the final enzyme concentration to 10 μM. Add indole stock solution to bring the final indole concentration to 2 mM. Completely immerse a 5 cm × 5 cm piece of pure white cotton fabric in the reaction solution. Place the reaction flask in a temperature-controlled magnetically stirred water bath at 37°C and stir gently. Add hydrogen peroxide in a single injection using a microsyringe to bring the final H2O2 concentration to 1 mM. Continue the reaction for 60 minutes.

[0202] After the reaction is complete, remove the dyed cotton fabric, rinse it twice with deionized water to remove loose pigment particles adhering to the surface, and let it air dry naturally.

[0203] Fastness testing was performed directly on the dyed fabric.

[0204] The fabric, after being dyed and dried once, was subjected to a dry rubbing fastness test directly according to the method specified in GB / T 3920-2008 "Textiles - Tests for Color Fastness - Color Fastness to Rubbing". The standard cotton lining fabric used for rubbing was 5cm × 5cm in size, the rubbing head applied a force of 9N, the reciprocating stroke was 100mm, and the cycle was repeated 10 times.

[0205] After the test, the staining grade of the standard cotton lining fabric for rubbing was assessed using the gray scale for staining assessment (GB / T 251). The results showed that the dry rubbing staining grade of the fabric was 3, and there was obvious blue staining on the lining fabric. When the fabric surface was lightly rubbed with a finger, obvious blue adhesion was visible on the fingertip.

[0206] Another small piece of the fabric was cut off and boiled in boiling water for 10 minutes. The liquid turned a distinct light blue, indicating that the indigo adhering to the fabric was easily dissolved and migrated in hot water.

[0207] Indigo-dyed fabrics without acid post-treatment have indigo pigment primarily adhering to the fiber surface in particulate form, with weak bonding between the pigment and the fiber. This makes them prone to detachment and migration under mechanical friction and hot water, resulting in a dry rubbing fastness grade of 3. This result serves as a reference for the dry rubbing fastness grade of 4 achieved in the fabric treated with ascorbic acid (a weakly acidic solution) in Example 3, demonstrating that post-treatment improves the colorfastness of the fabric.

[0208] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for preparing colored pigments based on bio-enzyme catalysis, characterized in that, Includes the following steps: S1. A reaction system is provided, the reaction system comprising a neuroglobin mutant with peroxidase activity, an indole substrate and hydrogen peroxide, wherein the indole substrate comprises a first substrate and a second substrate, the first substrate and the second substrate being the same or different indole compounds; S2. The neuroglobin mutant is mixed with the first part of the substrate in a reaction medium, and the first part of hydrogen peroxide is added to carry out a pre-reaction to generate a pre-reaction solution containing an indole radical intermediate; wherein, the molar amount of the first part of hydrogen peroxide is less than the theoretical molar amount of hydrogen peroxide required to completely convert all the indole compounds in the first part of the substrate into indigo dimers. S3. Add the remaining second portion of substrate and the second portion of hydrogen peroxide to the pre-reaction solution obtained in step S2, and continue the coupling reaction to generate indigo pigment products.

2. The method for preparing colored pigments based on bio-enzyme catalysis according to claim 1, characterized in that, In step S2, the molar amount of the first portion of hydrogen peroxide is 50% to 90% of the theoretical molar amount required to convert all the indole molecules in the first portion of the substrate into indole radicals.

3. The method for preparing colored pigments based on bio-enzyme catalysis according to claim 1, characterized in that, The molar ratio of the first portion of substrate to the second portion of substrate is 1:10 to 10:

1.

4. The method for preparing colored pigments based on bio-enzyme catalysis according to claim 1, characterized in that, The neuroglobin mutant is an A15C / H64D / F49Y triple mutant derived from human neuroglobin.

5. The method for preparing colored pigments based on bio-enzyme catalysis according to claim 1, characterized in that, The first substrate and the second substrate are each independently selected from one of indole, 4-chloroindole, 5-chloroindole, 6-chloroindole, 7-chloroindole, 4-nitroindole, 5-nitroindole and 6-bromoindole.

6. The method for preparing colored pigments based on bio-enzyme catalysis according to claim 1, characterized in that, It also includes S4: During the coupling reaction in step S3, the Lab color value of the reaction solution is collected in real time using a color sensor. When the detected Lab color value reaches the preset target color range, the coupling reaction is terminated by stopping the addition of hydrogen peroxide and lowering the reaction temperature.

7. The method for preparing colored pigments based on bio-enzyme catalysis according to claim 6, characterized in that, In step S4, when the detected Lab color value deviates from the preset target color range, a first substrate or a second substrate is added to the reaction system during step S3.

8. The method for preparing colored pigments based on bio-enzyme catalysis according to claim 1, characterized in that, It also includes the following steps: S5. A cellulose fiber fabric with indigo pigment particles attached after being subjected to the coupling reaction treatment in step S3. S6. Immerse the fabric in a weakly acidic aqueous solution containing ascorbic acid, so that the indigo pigment particles adhering to the surface of the fabric are reduced in situ to water-soluble leuco pigments, which penetrate into the internal pores of the cellulose fibers. S7. Remove the fabric from the solution and expose it to air or an oxygen-containing atmosphere to oxidize the leuco pigments that have penetrated into the fiber, regenerate indigo pigment crystals, and fix them inside the fiber.

9. The method for preparing colored pigments based on bio-enzyme catalysis according to claim 8, characterized in that, In step S6, the concentration of ascorbic acid in the weakly acidic aqueous solution is 0.5 g / L to 50 g / L, the pH value of the solution is 3.0 to 6.5, and the treatment temperature is 20°C to 60°C.

10. An integrated system for preparing colored pigments based on bio-enzyme catalysis, applicable to the method for preparing colored pigments based on bio-enzyme catalysis as described in any one of claims 1 to 9, characterized in that, include: A reaction vessel used to contain the reaction liquid; The feeding unit is used to add the substrate and hydrogen peroxide to the reaction vessel in steps or continuously; The detection unit includes a color sensor positioned facing the reaction solution for real-time acquisition of the Lab colorimetric value of the reaction solution; The control unit is communicatively connected to the detection unit and the feeding unit, and is used to receive the Lab colorimetric value and send a feeding instruction to the feeding unit based on the comparison result of the Lab colorimetric value and the preset target colorimetric range. The post-treatment unit is used to contain a weakly acidic aqueous solution containing ascorbic acid and to receive the fabric treated by the reaction vessel for immersion treatment in the weakly acidic aqueous solution.

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