Indirubin and preparation method therefor

Abstract

The present application relates to an indirubin preparation method comprising the steps of: performing primary culturing on an enzyme and E.coli so as to form a first culture medium such that the enzyme is expressed in the E.coli; mixing the primary culture medium with a substrate so as to form a second culture medium; performing secondary culturing on the second culture medium so as to form a third culture medium; and mixing the third culture medium with a solvent, and separating same to extract indirubin, wherein the substrate includes L.-tryptophan or indole, and the substrate is converted into indirubin by the E. coli and the enzyme.

Description

Indirubin and its preparation method

[0001] The present invention relates to indirubin and a method for producing the same.

[0002] Indirubin, an isomer of indigo, is red and was discovered from Danggui Longhui Wan, a traditional Chinese remedy. Indirubin is known to possess pharmacological activity against various diseases, and is specifically used as a treatment for various conditions, including leukemia, anti-inflammatory diseases, psoriasis, and skin rashes. Furthermore, indirubin derivatives produced through further chemical modification have been reported to exhibit very high pharmacological activity against various diseases.

[0003] Indirubin can be produced with indigo through the oxidation of indican, and unlike indigo, which is blue, it can be used as a red pigment. Fibers dyed with indirubin can take on a reddish tint when exposed to oxygen in the air.

[0004] Indirubin has significant utility in the pharmaceutical, dyeing, and food industries, attracting significant attention. However, due to a lack of clear synthetic pathways for indirubin or information regarding related genes and proteins, production is limited to quantities comparable to those produced as a byproduct during traditional indigo fermentation.

[0005] Specifically, the production pathway of indirubin, a by-product during indicotrienol fermentation, is not precisely known, but can be inferred based on the structures of the substrate, intermediates, and final product. First, during the fermentation process, indoxyl is released from indican by the plant's own enzyme (beta-glucosidase), and then another plant enzyme or fermentation microbial enzyme (monooxygenase or dioxygenase) converts it to isatin. Then, the two substances spontaneously combine (dimerize) in the presence of oxygen to produce indirubin. Therefore, it is presumed that the degree of indirubin production will vary depending on the natural abundance of the two enzyme sources, the relative enzyme activity ratio, and the amount of dissolved oxygen in the fermentation broth.

[0006] Korean Patent Publication No. 10-1021789, the background technology of this application, describes a method for producing indirubin from indican using wild-type E. coli.

[0007] The present invention is intended to solve the problems of the above-mentioned prior art, and provides a method for producing indirubin using E. coli and indirubin produced by E. coli.

[0008] However, the technical tasks to be achieved by the embodiments of the present invention are not limited to the technical tasks described above, and other technical tasks may exist.

[0009] As a technical means for achieving the above-described technical task, the first aspect of the present invention relates to a method for producing indirubin, comprising: a step of forming a first culture medium by first culturing the enzyme and the E. coli so that the enzyme is expressed in the E. coli; a step of forming a second culture medium by mixing the first culture medium and a substrate; a step of second culturing the second culture medium to form a third culture medium; and a step of mixing and separating the third culture medium and a solvent to extract indirubin; wherein the substrate includes L-tryptophan or indole, and the substrate is converted into indirubin by the E. coli and the enzyme.

[0010] According to one embodiment of the present invention, the first culturing step may include, but is not limited to, a step of seed culturing the E. coli and the enzyme and a step of multiplying the E. coli and the enzyme.

[0011] According to one embodiment of the present invention, the enzyme may include, but is not limited to, PmT4MO enzyme (Pseudomonas mendocina KR1 toluene-4-monooxygenase) represented by SEQ ID NO: 1, or Mafmo enzyme (flavin-containing monooxygenase from Methylophaga aminisulfidivorans).

[0012] According to one embodiment of the present invention, the E. coli may include, but is not limited to, Escherichia coli BL21 (DE3).

[0013] According to one embodiment of the present invention, the E. coli may include, but is not limited to, tryptophanase (tnaA), which is an endogenous E. coli gene.

[0014] According to one embodiment of the present invention, the primary culturing step may be performed by culturing the E. coli and the enzyme until the OD600 of the primary culture solution becomes 0.5 to 0.9, but is not limited thereto.

[0015] According to one embodiment of the present invention, the step of forming the secondary culture solution may further include, but is not limited to, a step of mixing a material selected from the group consisting of a surfactant, a pH adjusting material, a sulfur-containing material, and combinations thereof onto the primary culture solution.

[0016] According to one embodiment of the present invention, the surfactant may include, but is not limited to, a surfactant selected from the group consisting of polysorbate 80, polysorbate 60, polysorbate 40, polysorbate 20, and combinations thereof.

[0017] According to one embodiment of the present invention, the concentration of the surfactant in the secondary culture medium may be, but is not limited to, 0.1% to 10%.

[0018] According to one embodiment of the present invention, the pH adjusting substance may include, but is not limited to, a substance selected from the group consisting of HCl, H2SO4, NaOH, KOH, and combinations thereof.

[0019] According to one embodiment of the present invention, the sulfur-containing material may include, but is not limited to, one selected from the group consisting of FeSO4, Na2SO4, (NH4)2SO4, MgSO4, methionine, cysteine, and combinations thereof.

[0020] According to one embodiment of the present invention, the tertiary culture medium may include, but is not limited to, indigo and indirubin.

[0021] According to one embodiment of the present invention, the solvent can selectively extract indirubin, but is not limited thereto.

[0022] According to one embodiment of the present invention, the solvent may include, but is not limited to, a solvent selected from the group consisting of alcohols having 1 to 5 carbon atoms, ketones having 2 to 5 carbon atoms, DMSO, ethyl acetate, and combinations thereof.

[0023] In addition, the second aspect of the present invention relates to indirubin, which is manufactured by the method according to the first aspect.

[0024] The above-described problem-solving methods are merely exemplary and should not be construed as limiting the present invention. In addition to the exemplary embodiments described above, additional embodiments may be included in the drawings and detailed description of the invention.

[0025] It is generally known that indigo is produced concurrently with indirubin during the production of indirubin. The indirubin production method according to the present invention utilizes ethanol, which is easy to handle and can be used in various bio-industries, as an extraction solvent to selectively extract only indirubin from among indirubin and indigo, thereby producing indirubin at a high concentration.

[0026] In addition, the method for producing indirubin according to the present invention, that is, when producing indirubin using E. coli, can control and analyze the effects of various factors such as surfactant, pH, and sulfur.

[0027] In addition, indirubin manufactured through the above-described indirubin manufacturing method can be used in various fields such as clothing and pharmaceuticals.

[0028] In addition, the method for producing indirubin according to the present invention can produce indirubin using L-tryptophan, which has low toxicity and is easy to use industrially.

[0029] However, the effects that can be obtained from this center are not limited to the effects described above, and other effects may exist.

[0030] Figure 1 is a flowchart showing a method for manufacturing indirubin according to one embodiment of the present invention.

[0031] Figure 2 is a schematic diagram showing a method for manufacturing indirubin according to one embodiment of the present invention.

[0032] FIGS. 3a and 3b are photographs of indirubin manufactured by a method according to one embodiment of the present invention.

[0033] Figure 4 is a photograph of indirubin manufactured by a method according to one embodiment of the present invention.

[0034] Figure 5 is a photograph of indirubin manufactured by a method according to one embodiment of the present invention.

[0035] Figure 6 is a graph analyzing the production amount of indirubin by a method according to one embodiment of the present invention.

[0036] FIGS. 7A to 7F are photographs of indirubin manufactured by a method according to one embodiment of the present invention.

[0037] Figure 8 is a graph analyzing the production amount of indirubin by a method according to one embodiment of the present invention.

[0038] FIGS. 9A and 9B are photographs of indirubin manufactured by a method according to one embodiment of the present invention.

[0039] Figure 10 is a photograph of indirubin manufactured by a method according to one embodiment of the present invention.

[0040] FIG. 11a is a photograph of indirubin produced by a method according to one embodiment of the present invention, and FIGS. 11b to 11d are graphs analyzing the production amount of indirubin produced by a method according to one embodiment of the present invention.

[0041] FIG. 12a is a photograph of indirubin produced by a method according to one embodiment of the present invention, and FIGS. 12b to 12d are graphs analyzing the production amount of indirubin produced by a method according to one embodiment of the present invention.

[0042] FIG. 13a is a photograph of indirubin produced by a method according to one embodiment of the present invention, and FIGS. 13b to 13d are graphs analyzing the production amount of indirubin produced by a method according to one embodiment of the present invention.

[0043] FIG. 14a is a graph analyzing the production amount of indirubin by a method according to one embodiment of the present invention, and FIGS. 14b and 14c are photographs of indirubin produced by a method according to one embodiment of the present invention.

[0044] Below, with reference to the attached drawings, an embodiment of the present invention is described in detail so that a person having ordinary knowledge in the technical field to which the present invention pertains can easily carry out the present invention.

[0045] However, the present invention can be implemented in various different forms and is not limited to the embodiments described herein. In order to clearly explain the present invention in the drawings, parts irrelevant to the description have been omitted, and similar parts have been designated with similar drawing reference numerals throughout the specification.

[0046] Throughout this specification, when a part is said to be "connected" to another part, this includes not only cases where it is "directly connected" but also cases where it is "electrically connected" with another element in between.

[0047] Throughout this specification, when it is said that a member is located “on,” “above,” “upper,” “lower,” “lower” or “lower” another member, this includes not only cases where the member is in contact with the other member, but also cases where another member exists between the two members.

[0048] Throughout this specification, whenever a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise specifically stated.

[0049] The terms "about," "substantially," and the like, as used herein, are used to mean at or near the numerical value when manufacturing and material tolerances inherent to the meanings referred to are presented, and are used to prevent unscrupulous infringers from unfairly exploiting disclosures that contain precise or absolute numerical values ​​to aid understanding of the present disclosure. Furthermore, throughout the present disclosure, the terms "step of ~" or "step of ~" do not mean "step for ~."

[0050] Throughout this specification, the term "combination thereof" included in the expressions in the Makushi format means one or more mixtures or combinations selected from the group consisting of the components described in the expressions in the Makushi format, and means including one or more selected from the group consisting of said components.

[0051] Throughout this specification, references to “A and / or B” mean “A or B, or A and B.”

[0052] Hereinafter, indirubin and its manufacturing method will be described in detail with reference to implementation examples, examples, and drawings. However, the present invention is not limited to these implementation examples, examples, and drawings.

[0053] As a technical means for achieving the above-described technical task, the first aspect of the present invention relates to a method for producing indirubin, comprising: a step of forming a first culture medium by first culturing the enzyme and the E. coli so that the enzyme is expressed in the E. coli; a step of forming a second culture medium by mixing the first culture medium and a substrate; a step of second culturing the second culture medium to form a third culture medium; and a step of mixing and separating the third culture medium and a solvent to extract indirubin; wherein the substrate includes L-tryptophan or indole, and the substrate is converted into indirubin by the E. coli and the enzyme.

[0054] The method for producing indirubin according to the present invention converts a substrate into indirubin using E. coli, and can be referred to as a bio-indirubin production method since indirubin is produced biologically.

[0055] FIG. 1 is a flowchart showing a method for producing indirubin according to an embodiment of the present disclosure, and FIG. 2 is a schematic diagram showing a method for producing indirubin according to an embodiment of the present disclosure. As will be described later, T4mo in FIG. 2 refers to PmT4MO of the present disclosure. In addition, the arrow pointing from 3-hydroxyindole to indirubin refers to dimerization with 3-hydroxyindole and 7-hydroxyindole.

[0056] First, the enzyme and the E. coli are cultured in primary culture to form a primary culture solution so that the enzyme is expressed in E. coli.

[0057] According to one embodiment of the present invention, the primary culturing step may include, but is not limited to, a step of seed culturing the E. coli and the enzyme and a step of propagating the E. coli and the enzyme. In this case, the propagation culturing refers to a subculture.

[0058] According to one embodiment of the present invention, the E. coli may include, but is not limited to, Escherichia coli BL21 (DE3).

[0059] According to one embodiment of the present invention, the E. coli may include, but is not limited to, tryptophanase (tnaA), an endogenous enzyme of E. coli. The tnaA is an endogenous enzyme of E. coli that bioconverts tryptophan into indole.

[0060] Escherichia coli according to the present invention is a bacterium that can be found in large quantities in the intestines of warm-blooded animals, and can also be expressed as E. coli. The E. coli is known as a widely used specimen organism because it reproduces easily and is genetically simple. In addition, E. coli BL21 is a strain derived from strain B that lacks lon protease and ompT outer membrane protease, and is widely used for recombinant protein expression because it can improve the stability of expressed proteins. E. coli BL21 (DE3) according to the present invention means E. coli BL21 with DE3 (including T7 RNA polymerase) inserted into it.

[0061] Meanwhile, tnaA refers to an enzyme that functions as a catalyst for the reaction that converts tryptophan and water into indole, pyruvate, and ammonium ions. Referring to Figure 2, it can be confirmed that L-tryptophan is converted into indole by tnaA.

[0062] In this regard, when the E. coli and the enzyme are seed-cultured, the enzyme is incorporated into the E. coli, so that the E. coli contains tnaA and the enzyme. Subsequently, through the propagation culture, the E. coli containing the enzyme and tnaA can be propagated.

[0063] According to one embodiment of the present invention, the enzyme may include, but is not limited to, the PmT4MO enzyme (Pseudomonas mendocina KR1 toluene-4-monooxygenase) represented by SEQ ID NO: 1, or the Mafmo enzyme (flavin-containing monooxygenase from Methylophaga aminisulfidivorans). In this case, Pm in the description of the PmT4MO means that it is derived from Pseudomonas mendocina.

[0064] The above PmT4MO enzyme is an oxidase derived from a strain called Pseudomonas mendocina, and the Mafmo enzyme refers to an oxidase derived from a strain called Methylophaga aminisulfidivorans. At this time, the Pmt4mo enzyme has a size of approximately 4.3 kb, and the mafmo enzyme has a size of approximately 1.3 kb. In this regard, although the PmT4MO enzyme and the Mafmo enzyme are oxidases, there is a difference in the amount of indirubin produced when producing indirubin according to the present invention, which is due to the structural difference between the two enzymes.

[0065] A monooxygenase enzyme is an enzyme that catalyzes a reaction in which one oxygen atom of an oxygen molecule binds to a substrate (e.g., L-tryptophan) and the other oxygen atom is reduced to water. The above E. coli has tnaA built-in, and this means that the enzyme is additionally built-in during the step of forming the primary culture medium.

[0066] According to one embodiment of the present invention, the first culturing step is to determine the OD of the first culture solution. 600 The E. coli and the enzyme can be cultured until the concentration reaches 0.5 to 0.9, but is not limited thereto.

[0067] OD according to this document 600refers to the optical density for light in the 600 nm wavelength band. In this regard, optical density is a measure of the ability of a refractive medium or optical component to slow or retard the transmission of light, and the slower the speed of light passing through the medium, the higher the optical density.

[0068] According to one embodiment of the present invention, the seed culturing step may be performed at a temperature of 30°C to 40°C and a rotation speed of 100 rpm to 300 rpm for 14 to 20 hours, but is not limited thereto.

[0069] The above seed culturing step may include a step of adding and culturing E. coli and enzymes in a volume corresponding to 1% of the volume of a medium (eg, LB medium).

[0070] The above-mentioned proliferation culturing step may be, but is not limited to, mixing the seed-cultured culture solution with a medium (LB medium, for example) having a volume corresponding to 50 to 100 times the seed-cultured culture solution and culturing the mixture.

[0071] Next, the primary culture medium and substrate are mixed to form a secondary culture medium. At this time, the OD of the primary culture medium mixed with the substrate 600 is 0.5 to 0.9.

[0072] According to one embodiment of the present invention, the substrate may be, but is not limited to, L-tryptophan or indole.

[0073] Referring to FIG. 2, the method for producing indirubin according to the present invention converts L-tryptophan into indole through tnaA of the E. coli, and then the indole is converted into hydroxyindole by an enzyme (PmT4MO or Mafmo) of the E. coli. In this regard, the secondary culture means a state in which the substrate and the primary culture are simply mixed. As will be described later, by secondary culturing the secondary culture under given conditions, the E. coli of the primary culture (including tnaA and the enzyme) can convert the substrate.

[0074] According to one embodiment of the present invention, the step of forming the secondary culture solution may further include, but is not limited to, a step of mixing a material selected from the group consisting of a surfactant, a pH adjusting material, a sulfur-containing material, and combinations thereof onto the primary culture solution.

[0075] At this time, when the secondary culture solution is cultured, the substrate conversion reaction by the E. coli can be controlled by the surfactant, pH control substance, and sulfur-containing substance.

[0076] According to one embodiment of the present invention, the surfactant may include, but is not limited to, a surfactant selected from the group consisting of polysorbate 80, polysorbate 60, polysorbate 40, polysorbate 20, and combinations thereof.

[0077] The above polysorbate is a type of nonionic surfactant, and polysorbate 80 may also be called tween 80, polysorbate 60 may also be called tween 60, polysorbate 40 may also be called tween 40, and polysorbate 20 may also be called tween 20.

[0078] Preferably, the surfactant may be, but is not limited to, tween 80 (polysorbate 80). In this regard, the surfactant may include a substance that exhibits similar activity in the indole-indirubin metabolism as polysorbate 80.

[0079] According to one embodiment of the present invention, the concentration of the surfactant in the secondary culture medium may be, but is not limited to, 0.1% to 10%. In this case, the concentration of the surfactant may be defined as the volume of the surfactant relative to the volume of the secondary culture medium.

[0080] As the amount of surfactant added to the secondary culture medium increases, the amount of indirubin produced may also increase.

[0081] When a surfactant is added, the movement of the substrate or product on the cell membrane becomes smooth, and it was confirmed that more blue indigo was produced in the experimental group without tween80. That is, when PmT4MO is used, indirubin is produced as an early metabolic product inside the cell, and indirubin is secreted outside the cell by the surfactant (tween80), and accordingly, indirubin saturated inside the cell is discharged outside the cell, thereby increasing the production of indirubin in the E. coli. However, when the surfactant is not added, indirubin is not secreted outside the cell after being produced, but rather indigo is produced more predominantly and then secreted, so the production of indirubin can increase due to the surfactant.

[0082] According to one embodiment of the present invention, the pH adjusting substance may include, but is not limited to, a substance selected from the group consisting of HCl, H2SO4, NaOH, KOH, and combinations thereof.

[0083] In this regard, when the pH adjusting substance is HCl, the production amount of indirubin may increase as the pH of the secondary culture medium decreases, but when the pH adjusting substance is H2SO4, the production amount of indirubin is the highest when the pH is 6, and the production amount of indirubin may vary when the pH is lower or higher than 6. In this regard, the production amount of indirubin is higher when the pH adjusting substance is H2SO4 than when the pH adjusting substance is HCl.

[0084] According to one embodiment of the present invention, the sulfur-containing material may include, but is not limited to, one selected from the group consisting of FeSO4, Na2SO4, (NH4)2SO4, MgSO4, methionine, cysteine, and combinations thereof.

[0085] When the above secondary culture medium contains sulfur, i.e., when H2SO4 is used as a pH adjusting substance or when the above sulfur-containing substance is included, the production of indirubin can increase.

[0086] Specifically, indirubin can be produced by dimerization of 7-hydroxyindole and 3-hydroxyindole. At this time, when the two 3-hydroxyindoles are dimerized, indigo is formed. The unshared electron pair contained in sulfur attacks the 7th carbon position of 3-hydroxyindole, and the sulfur-containing residue in the sulfur-containing substance combines with 3-hydroxyindole, and the proton of the hydroxyl group in 7-hydroxyindole is removed and O - Indirubin is formed when the sulfur-containing substance attacks the carbon of the residue bonded to 3-hydroxyindole while forming a bond with the carbon atom. That is, the dimerization that occurs between two 3-hydroxyindoles is inhibited by the sulfur-containing substance, resulting in the production of more indirubin.

[0087] Meanwhile, there is a pH optimized for activity for each enzyme, and the pH regulator is for setting a pH suitable for the enzyme.

[0088] According to one embodiment of the present invention, the secondary culture solution may additionally contain IPTG, but is not limited thereto.

[0089] The above IPTG refers to isopropyl β-D-1-thiogalactopyranoside (IPTG), a reagent used in molecular biology and biochemistry.

[0090] Next, the secondary culture is subcultured to form a tertiary culture.

[0091] As described above, the secondary culture medium may be a mixture of the substrate and the primary culture medium, and may additionally contain a material selected from the group consisting of the surfactant, the pH adjusting material, the sulfur-containing material, and combinations thereof.

[0092] According to one embodiment of the present invention, the tertiary culture medium may include, but is not limited to, indigo and indirubin.

[0093] Referring to Figure 2, the indole can be converted into 3-hydroxyindole or 7-hydroxyindole by the enzyme (PmT4MO or Mafmo). At this time, the hydroxyindole can be dimerized by oxidase and converted into indigo or indirubin.

[0094] That is, the above tertiary culture medium can contain indigo and indirubin simultaneously.

[0095] Next, the above-mentioned third culture medium and solvent are mixed and separated to extract indirubin.

[0096] According to one embodiment of the present invention, the solvent can selectively extract indirubin, but is not limited thereto.

[0097] According to one embodiment of the present invention, the solvent may include, but is not limited to, a solvent selected from the group consisting of an alcohol having 1 to 5 carbon atoms, a ketone having 2 to 5 carbon atoms, DMSO, ethyl acetate, and combinations thereof. Preferably, the solvent may include ethanol.

[0098] Since the above 3rd culture medium is a mixture of indigo and indirubin, it is necessary to select a solvent that selectively extracts only indirubin. As a result of quantifying the absorbance of the solvents using a spectrophotometer at a maximum absorbance wavelength of 550 nm, it was confirmed that ethanol showed an absorbance of 0.712, acetone showed an absorbance of 0.602, methanol showed an absorbance of 0.648, EA showed an absorbance of 0.341, and DMSO showed an absorbance of 0.681. In other words, it can be confirmed that ethanol among the above solvents is suitable for extracting indirubin.

[0099] When the above 3rd culture medium and the above solvent are mixed, only the indirubin can be selectively separated through the difference in the solubility of indigo and indirubin in the solvent.

[0100] In addition, the second aspect of the present invention relates to indirubin, which is manufactured by the method according to the first aspect.

[0101] The present invention will be described in more detail through the following examples; however, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0102] [Example]

[0103] First, seed culture was performed by adding 1% PmT4MO to 3 mL of LB medium in an incubation at 37°C and 200 rpm. Then, after 16 to 18 hours, 100 mL of LB medium was added to a flask with a 500 mL baffle, and 1 to 2 mL of the seed culture solution was added and cultured. Then, after about 4 hours, the OD of the culture was measured using a spectrophotometer. 600 After adjusting to 0.6 to 0.8, the substrate tryptophan was added to 10 mM. In addition, 7 mL of a surfactant (tween80) was added to make up 7% of the culture solution, sulfuric acid was added as a pH adjuster to adjust the pH to 6, 0.1 mM IPTG and 3 mM of a sulfur-containing substance (iron sulfide) were added, and cultured at 30°C and 200 rpm. After 24 hours, the ethanol and culture solution were placed in a 50 mL tube at a 1:1 ratio and centrifuged in a centrifuge to obtain indirubin from the supernatant excluding the downed cells.

[0104] [Comparative example]

[0105] Same as the above example, but Mafmo enzyme was used instead of PmT4MO enzyme, and the concentration of surfactant, amount and type of pH adjusting substance, type of sulfur-containing substance, and solvent used for extracting indirubin were changed.

[0106] Figures 3a and 3b are photographs of indirubin manufactured by a method according to an example and comparative example of the present invention. Specifically, Figure 3a represents a solution cultured with PmT4MO added and a solution cultured with Mafmo added, and Figure 3b is a photograph when the extraction solution was changed to NaOH, DMSO, and EA.

[0107] Referring to Figure 3, when producing indigo and indirubin using an oxidase enzyme, it can be confirmed that the solution using the PmT4MO enzyme shows a red color (i.e., more indirubin was extracted) and the solution using the Mafmo enzyme shows a blue color (i.e., more indigo was extracted) even when extracted using the same method.

[0108] Additionally, it can be confirmed that the reddest color is observed when extracting indirubin with DMSO.

[0109] [Experimental Example 1]

[0110] Indirubin was manufactured by adjusting the concentration of Tween80, a surfactant, to 1%, 3%, 5%, and 7%.

[0111] FIGS. 4, 5, and 7a to 7f are photographs of indirubin produced by a method according to an embodiment of the present disclosure, and FIG. 6 is a graph analyzing the production amount of indirubin by a method according to an embodiment of the present disclosure. Specifically, the left side of FIGS. 7a and 7b is a culture solution without a surfactant added, the right side is a culture solution with a surfactant added, and the left side of FIG. 7c is a culture solution without a surfactant added, indirubin extracted with water or ethanol, and the right side is a culture solution with a surfactant added, indirubin extracted with water or ethanol.

[0112] Referring to FIGS. 4 to 7f, the greater the amount of added surfactant (i.e., the greater the concentration of surfactant in the culture medium), the more red the culture medium becomes, which means that more indirubin is produced.

[0113] Additionally, it can be confirmed that when cultured without adding a surfactant, less indirubin is produced, but when cultured with the addition of a surfactant, more indirubin is produced.

[0114] [Experimental Example 2]

[0115] The amount of indirubin produced according to the culture time was compared.

[0116] Fig. 8 is a graph analyzing the production amount of indirubin by a method according to one embodiment of the present invention. Referring to Fig. 8, 1.96 mM of indirubin was produced 24 hours after culturing, and it can be confirmed that the production amount of indirubin decreases if the culturing is longer or shorter. In other words, 24 hours is an appropriate culturing time for indirubin production.

[0117] [Experimental Example 3]

[0118] When extracting the cultured broth, the solvent was changed to ethanol, acetone, methanol, and DMSO.

[0119] Figures 9a, 9b, and 10 are photographs of indirubin manufactured by a method according to one embodiment of the present invention. In this regard, the solution placed on the far right in ethanol of Figure 9a is PmT4MO (10 mM indole), and 0.276, 0.223, 0.640, and 0.526 placed at the bottom in DMSO represent the absorbance of indirubin when DMSO is used as a solvent.

[0120] Referring to FIGS. 9a and 9b, it can be seen that the color is generally redder when the PmT4MO enzyme is used than when the Mafmo enzyme is used, and the color is the reddest when extracted with ethanol, which confirms that the PmT4MO enzyme must be used and extracted with ethanol to produce indirubin.

[0121] Additionally, it can be confirmed that the amount of indirubin produced is relatively small when indole is used instead of tryptophan as a substrate.

[0122] [Experimental Example 4]

[0123] Fig. 11a is a photograph of indigo produced by a method according to one embodiment of the present invention, and Figs. 11b to 11d are graphs analyzing the production amount of indirubin by a method according to one embodiment of the present invention. Specifically, the left side of Fig. 11a shows indigo extracted with ethanol, and the right side of Fig. 11a shows indigo extracted with DMSO.

[0124] When DMSO is used as an extraction solvent, both indigo and indirubin are extracted well, but when ethanol is used, indirubin is easy to extract, but indigo is difficult to extract. That is, when we checked how much indigo and indirubin were produced from the substrate (tryptophan) using ethanol and DMSO as extraction solvents, approximately 29% was converted with ethanol and approximately 36% with DMSO, but it was confirmed that more indigo was extracted with DMSO compared to ethanol.

[0125] Additionally, it can be confirmed that when the culture temperature is higher (for example, 30°C) when producing indirubin, more indirubin is produced.

[0126] [Experimental Example 5]

[0127] FIG. 12a is a photograph of indirubin produced by a method according to an embodiment of the present disclosure, and FIGS. 12b to 12d are graphs analyzing the amount of indirubin produced by a method according to an embodiment of the present disclosure, and FIG. 13a is a photograph of indirubin produced by a method according to an embodiment of the present disclosure, and FIGS. 13b to 13d are graphs analyzing the amount of indirubin produced by a method according to an embodiment of the present disclosure. Specifically, FIG. 12 uses HCl as a pH adjusting substance, and FIG. 13 uses H2SO4, and when the pH is 7 or higher, NaOH is added to adjust the pH.

[0128] Referring to Figure 12, it can be confirmed that more indirubin is produced in an acidic pH environment, but the highest production amount is observed when the pH is 4.

[0129] In addition, since the sulfuric acid used in Fig. 13 is a stronger acid than hydrochloric acid, it is expected to be more toxic to cells than hydrochloric acid when added to the culture medium, and it can be confirmed that a smaller amount of indirubin is produced at pH 4 using H2SO4 than at pH 4 using HCl. However, it can be confirmed that more indirubin is produced when the pH is 6 due to the influence of sulfur.

[0130] [Experimental Example 6]

[0131] FIG. 14a is a graph analyzing the production amount of indirubin by a method according to one embodiment of the present invention, and FIGS. 14b and 14c are photographs of indirubin produced by a method according to one embodiment of the present invention.

[0132] When cultured with the addition of sulfur-containing substances, it can be confirmed that more indirubin is produced compared to the control group (cultured without sulfur-containing substances), and in particular, a large amount of indirubin is produced when FeSO4 or methionine is used.

[0133] The above description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single entity may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0134] The scope of the present invention is indicated by the claims described below rather than the detailed description above, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. A step of forming a primary culture by first culturing the enzyme and the E. coli so that the enzyme is expressed in E. coli; A step of forming a secondary culture medium by mixing the primary culture medium and substrate; A step of forming a tertiary culture by culturing the secondary culture solution; and A step of mixing and separating the above 3rd culture medium and solvent to extract indirubin; Including, The above substrate contains L-tryptophan or indole, The substrate is converted into indirubin by the above E. coli and the above enzyme. Method for producing indirubin.

2. In paragraph 1, A method for producing indirubin, wherein the first culturing step includes a step of seed culturing the E. coli and the enzyme and a step of propagating the E. coli and the enzyme.

3. In paragraph 1, A method for producing indirubin, wherein the enzyme comprises PmT4MO enzyme (Pseudomonas mendocina KR1 toluene-4-monooxygenase) represented by sequence number 1, or Mafmo enzyme (flavin-containing monooxygenase from Methylophaga aminisulfidivorans).

4. In paragraph 1, A method for producing indirubin, wherein the above E. coli comprises Escherichia coli BL21 (DE3).

5. In paragraph 4, A method for producing indirubin, wherein the above E. coli contains tryptophanase (tnaA), which is an endogenous E. coli gene.

6. In paragraph 1, The above primary culturing step is the OD of the primary culture solution 600 A method for producing indirubin, comprising culturing the E. coli and the enzyme until the pH reaches 0.5 to 0.

9.

7. In paragraph 1, A method for producing indirubin, wherein the step of forming the secondary culture solution further includes the step of mixing a substance selected from the group consisting of a surfactant, a pH adjusting substance, a sulfur-containing substance, and combinations thereof into the primary culture solution.

8. In paragraph 7, A method for producing indirubin, wherein the surfactant comprises a surfactant selected from the group consisting of polysorbate 80, polysorbate 60, polysorbate 40, polysorbate 20, and combinations thereof.

9. In paragraph 8, A method for producing indirubin, wherein the concentration of the surfactant in the secondary culture medium is 0.1% to 10%.

10. In paragraph 7, A method for producing indirubin, wherein the pH adjusting substance comprises a substance selected from the group consisting of HCl, H2SO4, NaOH, KOH, and combinations thereof.

11. In paragraph 7, A method for producing indirubin, wherein the sulfur-containing material comprises one selected from the group consisting of FeSO4, Na2SO4, (NH4)2SO4, MgSO4, methionine, cysteine, and combinations thereof.

12. In paragraph 1, A method for producing indirubin, wherein the above-mentioned third culture medium contains indigo and indirubin.

13. In paragraph 12, A method for producing indirubin, wherein the above solvent selectively extracts indirubin.

14. In paragraph 1, A method for producing indirubin, wherein the solvent comprises a solvent selected from the group consisting of alcohols having 1 to 5 carbon atoms, ketones having 2 to 5 carbon atoms, DMSO, ethyl acetate, and combinations thereof.

15. Indirubin, manufactured by a method according to any one of claims 1 to 14.

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