Method for preparing indirubin

Abstract

The present application relates to a method for preparing indirubin, comprising the steps of: forming a primary culture medium by primarily culturing an enzyme and Escherichia coli so that the enzyme is expressed in the Escherichia coli; adding isatin to the primary culture medium so as to form a secondary culture medium; secondarily culturing the secondary culture medium so as to form a tertiary culture medium; and mixing the tertiary culture medium and a solvent and performing separation so as to extract indirubin, wherein the isatin is converted into indirubin by using the Escherichia coli and the enzyme.

Description

Method for manufacturing indirubin

[0001] The present invention relates to a method for manufacturing indirubin.

[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 prior art described above, and provides a method for producing indirubin through E. coli by adding isatin obtained from indigo.

[0008] In addition, indirubin manufactured by the above manufacturing method is provided.

[0009] In addition, a pharmaceutical composition comprising the above indirubin is provided.

[0010] 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.

[0011] As a technical means for achieving the above-described technical task, the first aspect of the present invention provides a method for producing indirubin, comprising the steps of: forming a first culture solution by first culturing the enzyme and the E. coli so that the enzyme is expressed in the E. coli; forming a second culture solution by adding isatin to the first culture solution; forming a third culture solution by second culturing the second culture solution; and mixing and separating the third culture solution and a solvent to extract indirubin, wherein the isatin is converted into indirubin by the E. coli and the enzyme.

[0012] According to one embodiment of the present invention, in the step of forming the secondary culture medium, a substrate may be additionally mixed into the primary culture medium, but is not limited thereto.

[0013] According to one embodiment of the present invention, the substrate may include, but is not limited to, L-tryptophan or indole.

[0014] According to one embodiment of the present invention, the substrate may be converted into indirubin by the E. coli and the enzyme, but is not limited thereto.

[0015] According to one embodiment of the present invention, the isatin may be produced by decomposing indigo with UV, but is not limited thereto.

[0016] According to one embodiment of the present invention, the indigo may be extracted from jeans, but is not limited thereto.

[0017] 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.

[0018] 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).

[0019] According to one embodiment of the present invention, the E. coli may include, but is not limited to, Escherichia coli BL21 (DE3).

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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%.

[0025] 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.

[0026] 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.

[0027] According to one embodiment of the present invention, the tertiary culture medium may include, but is not limited to, indigo and indirubin.

[0028] According to one embodiment of the present invention, the solvent can selectively extract indirubin, but is not limited thereto.

[0029] 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.

[0030] In addition, the second aspect of the present invention provides indirubin, manufactured by the method according to the first aspect of the present invention.

[0031] In addition, the third aspect of the present invention provides a pharmaceutical composition comprising indirubin according to the second aspect of the present invention.

[0032] 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.

[0033] The method for producing indirubin according to the present invention can efficiently produce indirubin using E. coli, and can produce a larger amount of indirubin than the existing method by using isatin obtained by decomposing indigo extracted from jeans with UV as an additional substrate, and this is an eco-friendly and economical manufacturing method that can produce indirubin, a high value-added material, by recycling jeans, which are industrial waste.

[0034] In addition, the method for producing indirubin according to the present invention can increase the amount of indirubin produced and decrease the amount of indigo produced by controlling the amount of isatin regardless of whether a surfactant (e.g., Tween 80) is added.

[0035] In addition, the method for producing indirubin according to the present invention can produce indirubin at a high concentration by using ethanol, which can be used in various bio-industries and is easy to handle, as an extraction solvent to selectively extract only indirubin among indirubin and indigo.

[0036] 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.

[0037] In addition, indirubin manufactured through the above-described indirubin manufacturing method can be used in various fields such as clothing and pharmaceuticals.

[0038] 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.

[0039] However, the effects that can be obtained from this center are not limited to the effects described above, and other effects may exist.

[0040] Figure 1 is a flowchart showing a method for manufacturing indirubin according to one embodiment of the present invention.

[0041] Figure 2 is a schematic diagram showing a method for manufacturing indirubin according to one embodiment of the present invention.

[0042] Figure 3 shows the results of comparing the amount of indirubin and indigo produced when indirubin was manufactured by adding Tween 80 according to one experimental example of the present invention.

[0043] Figure 4 shows the results of comparing the production amounts of indirubin and indigo when indirubin is manufactured without adding Tween 80 according to an experimental example of the present invention.

[0044] Figure 5 shows the results of comparing the amount of indirubin produced according to the tryptophan content when the concentration of isatin is fixed according to one experimental example of the present invention.

[0045] Figure 6 shows the results of measuring the amount of indirubin produced by adding isatin, which is formed by decomposing synthetic indigo with UV (254 nm) according to an experimental example of the present invention, to a culture medium.

[0046] Figure 7 shows the results of measuring the amount of indirubin produced by adding isatin, which is formed by decomposing indigo extracted from jeans under UV (254 nm), to a culture medium.

[0047] 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.

[0048] However, the present invention can be implemented in various different forms and is not limited to the embodiments described herein. Furthermore, 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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 ~."

[0053] 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.

[0054] Throughout this specification, references to “A and / or B” mean “A or B, or A and B.”

[0055] Hereinafter, a method for manufacturing indirubin 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.

[0056] As a technical means for achieving the above-described technical task, the first aspect of the present invention provides a method for producing indirubin, comprising the steps of: forming a first culture solution by first culturing the enzyme and the E. coli so that the enzyme is expressed in the E. coli; forming a second culture solution by adding isatin to the first culture solution; forming a third culture solution by second culturing the second culture solution; and mixing and separating the third culture solution and a solvent to extract indirubin, wherein the isatin is converted into indirubin by the E. coli and the enzyme.

[0057] The method for manufacturing indirubin according to the present invention converts isatin into indirubin using E. coli, and can be referred to as a bio-indirubin manufacturing method since indirubin is manufactured using a biological method.

[0058] Fig. 1 is a flowchart showing a method for producing indirubin according to one embodiment of the present disclosure, and Fig. 2 is a schematic diagram showing a method for producing indirubin according to one embodiment of the present disclosure. In this regard, T4mo in Fig. 2 refers to PmT4MO of the present disclosure. In addition, the arrow pointing from 3-hydroxyindole to indirubin refers to the dimerization of 3-hydroxyindole and 2-hydroxyindole, and the arrow pointing from isatin to indirubin refers to the dimerization of isatin and 2-hydroxyindole.

[0059] 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.

[0060] 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 subculturing the E. coli and the enzyme.

[0061] According to one embodiment of the present invention, the E. coli may include, but is not limited to, Escherichia coli BL21 (DE3).

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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).

[0067] In the description of the above PmT4MO, Pm means derived from Pseudomonas mendocina. That is, the PmT4MO enzyme is an oxidase derived from a strain called Pseudomonas mendocina, and the Mafmo enzyme is an oxidase derived from a strain called Methylophaga aminisulfidivorans. At this time, the Pmt4mo enzyme has a size of about 4.3 kb, and the mafmo enzyme has a size of about 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.

[0068] 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.

[0069] 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.

[0070] OD according to this invention 600 refers 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.

[0071] 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.

[0072] 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).

[0073] 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.

[0074] Next, isatin is added to the primary culture medium to form a secondary culture medium. At this time, the OD of the primary culture medium mixed with isatin 600 is 0.5 to 0.9.

[0075] Referring to Fig. 2, in the method for producing indirubin according to the present invention, it can be confirmed that isatin is added. By adding isatin, the production of indigo can be suppressed, thereby significantly increasing the production amount of indirubin, and thus, selective production of the desired product, indirubin, can be enabled.

[0076] According to one embodiment of the present invention, the isatin may be produced by decomposing indigo with UV, but is not limited thereto.

[0077] By using isatin produced by decomposing indigo with UV, it is possible to reduce costs by producing isatin directly from inexpensive indigo raw materials without having to separately synthesize or purchase isatin, and by converting inexpensive indigo into a precursor of indirubin, a high value-added substance, it is possible to increase the economic value of the overall process.

[0078] According to one embodiment of the present invention, the indigo may be extracted from jeans, but is not limited thereto.

[0079] By extracting indigo from jeans, we can reduce environmental pollution through recycling indigo-containing waste, such as jeans, and minimize the production of harmful byproducts that can arise during chemical synthesis. This serves as a good example of a circular economy model and can contribute to enhancing the sustainability of the industry.

[0080] According to one embodiment of the present invention, in the step of forming the secondary culture medium, a substrate may be additionally mixed into the primary culture medium, but is not limited thereto.

[0081] According to one embodiment of the present invention, the substrate may include, but is not limited to, L-tryptophan or indole.

[0082] According to one embodiment of the present invention, the substrate may be converted into indirubin by the E. coli and the enzyme, but is not limited thereto.

[0083] 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, isatin, and the primary culture are simply mixed. As 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] In the manufacturing method according to the present invention, when the surfactant is added, the production amounts of indirubin and indigo can be controlled depending on the ratio of the tryptophan and isatin. Referring to Experimental Example 1 described below, when 7% of Tween 80 was added, it was confirmed that when tryptophan and isatin were added at a ratio of 9:1, the production amount of indirubin increased and the production amount of indigo decreased compared to when only tryptophan was added. In addition, as the ratio of isatin increased (tryptophan and isatin ratios of 7:3 and 5:5), the production amount of indirubin decreased, but at the same time, the production amount of indigo also greatly decreased, confirming that indirubin can be produced more selectively.

[0091] In addition, when the above surfactant is not added, the amount of indirubin produced can be controlled depending on the content of isatin. Referring to Experimental Example 2 described below, it was confirmed that as the amount of isatin increased, the content of indirubin increased and the content of indigo decreased. When the production amounts were compared, it was confirmed that adding 2 mM of isatin produced the most indirubin. In addition, when the content of isatin was added more than 2 mM, the amount of indirubin produced itself decreased, but at the same time, the amount of indigo produced also greatly decreased, confirming that indirubin can be produced more selectively.

[0092] In this way, in the manufacturing method according to the present invention, the amount of indirubin produced can be controlled by appropriately adjusting the amount of tryptophan and isatin added depending on whether a surfactant is added or not, thereby achieving the best efficiency in various production environments by identifying the optimal conditions when a surfactant is used and when it is not used, and by adjusting the ratio of tryptophan and isatin, the purity and yield of the final product can be maintained at a constant level, thereby ensuring consistency in product quality, and by controlling the amount of indigo produced as a by-product, the efficiency of the overall process can be increased.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] Specifically, indirubin can be produced by dimerization of 2-hydroxyindole and 3-hydroxyindole. At this time, when the two 3-hydroxyindoles dimerize, indigo is formed. When the unshared electron pair contained in sulfur attacks the 7th carbon position of 3-hydroxyindole, the sulfur-containing residue in the sulfur-containing substance bonds with 3-hydroxyindole, and when the proton of the hydroxyl group in 2-hydroxyindole is removed and the O- group forms a bond with carbon, it attacks the carbon of the sulfur-containing residue bonded to 3-hydroxyindole, thereby forming indirubin. That is, the dimerization that occurs between the two 3-hydroxyindoles is suppressed by the sulfur-containing substance, and as a result, more indirubin is produced.

[0098] Meanwhile, there is a pH optimized for activity for each enzyme, and the pH regulator is for setting a pH suitable for the enzyme.

[0099] According to one embodiment of the present invention, the secondary culture solution may additionally contain IPTG, but is not limited thereto.

[0100] The above IPTG refers to isopropyl β-D-1-thiogalactopyranoside (IPTG), a reagent used in molecular biology and biochemistry.

[0101] Next, the secondary culture is subcultured to form a tertiary culture.

[0102] 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.

[0103] According to one embodiment of the present invention, the tertiary culture medium may include, but is not limited to, indigo and indirubin.

[0104] Referring to Figure 2, the indole can be converted into 3-hydroxyindole or 2-hydroxyindole by the enzyme (PmT4MO or Mafmo). At this time, the hydroxyindole can be dimerized by oxidase and converted into indigo or indirubin.

[0105] That is, the above tertiary culture medium can contain indigo and indirubin simultaneously.

[0106] Next, the above-mentioned third culture medium and solvent are mixed and separated to extract indirubin.

[0107] According to one embodiment of the present invention, the solvent can selectively extract indirubin, but is not limited thereto.

[0108] 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.

[0109] 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. That is, it can be confirmed that ethanol among the above solvents is suitable for extracting indirubin.

[0110] 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.

[0111]

[0112] In addition, the second aspect of the present invention provides indirubin, manufactured by the method according to the first aspect of the present invention.

[0113] Regarding indirubin according to the second aspect of the present application, detailed descriptions of parts overlapping with the first aspect of the present application have been omitted, but even if the descriptions have been omitted, the contents described in the first aspect of the present application may be equally applied to the second aspect of the present application.

[0114] In addition, the third aspect of the present invention provides a pharmaceutical composition comprising indirubin according to the second aspect of the present invention.

[0115] Regarding the pharmaceutical composition according to the third aspect of the present invention, detailed descriptions of parts overlapping with the first aspect and / or the second aspect of the present invention are omitted, but even if the descriptions are omitted, the contents described in the first aspect and / or the second aspect of the present invention can be equally applied to the third aspect of the present invention.

[0116] 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.

[0117]

[0118] [Comparative Example 1] Tween 80, 10 mM tryptophan

[0119] First, seed culture was performed by adding 1% PmT4MO to 3 mL of LB medium in an incubation at 37℃ 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 mL to 2 mL of the seed culture solution was added and cultured. Then, after about 4 hours, the OD600 of the culture solution was adjusted to 0.6 to 0.8 using a spectrophotometer, and then 10 mM of substrate tryptophan was added. 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, and 0.1 mM IPTG and 3 mM of a sulfur-containing substance (iron sulfide) were added and cultured at 30℃ and 200 rpm. Then, after 24 hours, the ethanol and culture medium are placed in a 50 mL tube at a ratio of 1:1 and centrifuged using a centrifuge to obtain indirubin from the supernatant excluding the downed cells.

[0120] [Comparative Example 2] Tryptophan 10 mM

[0121] It was manufactured in the same manner as Comparative Example 1, but without adding tween 80.

[0122] [Comparative Example 3] Tryptophan 3mM

[0123] It was manufactured in the same manner as Comparative Example 2, but with the addition of 3 mM tryptophan.

[0124]

[0125] [Example 1-1] Tween 80, 9 mM tryptophan, and 1 mM isatin

[0126] It was manufactured in the same manner as Comparative Example 1, but instead of adding 10 mM tryptophan, it was manufactured by adding 9 mM tryptophan and 1 mM isatin.

[0127] [Example 1-2] Tween 80, 7 mM tryptophan, and 3 mM isatin

[0128] It was manufactured in the same manner as Comparative Example 1, but instead of adding 10 mM tryptophan, it was manufactured by adding 7 mM tryptophan and 3 mM isatin.

[0129] [Example 1-3] Tween 80, 5 mM tryptophan, and 5 mM isatin

[0130] It was manufactured in the same manner as Comparative Example 1, but instead of adding 10 mM tryptophan, it was manufactured by adding 5 mM tryptophan and 5 mM isatin.

[0131] [Example 2-1] Tryptophan 3 mM and isatin 1 mM

[0132] It was manufactured in the same manner as Comparative Example 2, but instead of adding 10 mM tryptophan, it was manufactured by adding 3 mM tryptophan and 1 mM isatin.

[0133] [Example 2-2] Tryptophan 3 mM and isatin 2 mM

[0134] It was manufactured in the same manner as Comparative Example 2, but instead of adding 10 mM tryptophan, it was manufactured by adding 3 mM tryptophan and 2 mM isatin.

[0135] [Example 2-3] Tryptophan 3 mM and isatin 1 mM

[0136] It was manufactured in the same manner as Comparative Example 2, but instead of adding 10 mM tryptophan, it was manufactured by adding 3 mM tryptophan and 3 mM isatin.

[0137] [Example 2-4] Tryptophan 3 mM and isatin 4 mM

[0138] It was manufactured in the same manner as Comparative Example 2, but instead of adding 10 mM tryptophan, it was manufactured by adding 3 mM tryptophan and 4 mM isatin.

[0139] [Example 2-5] Tryptophan 3 mM and isatin 5 mM

[0140] It was manufactured in the same manner as Comparative Example 2, but instead of adding 10 mM tryptophan, it was manufactured by adding 3 mM tryptophan and 5 mM isatin.

[0141] [Example 2-6] Tryptophan 3 mM and isatin 6 mM

[0142] It was manufactured in the same manner as Comparative Example 2, but instead of adding 10 mM tryptophan, it was manufactured by adding 3 mM tryptophan and 6 mM isatin.

[0143] [Example 3-1] 1 mM tryptophan and 2 mM isatin

[0144] It was manufactured in the same manner as Comparative Example 2, but instead of adding 10 mM tryptophan, it was manufactured by adding 1 mM tryptophan and 2 mM isatin.

[0145] [Example 3-2] Tryptophan 2 mM and isatin 2 mM

[0146] It was manufactured in the same manner as Comparative Example 2, but instead of adding 10 mM tryptophan, it was manufactured by adding 2 mM tryptophan and 2 mM isatin.

[0147] [Example 3-3] Tryptophan 3 mM and isatin 2 mM

[0148] It was manufactured in the same manner as Comparative Example 2, but instead of adding 10 mM tryptophan, it was manufactured by adding 3 mM tryptophan and 2 mM isatin.

[0149] [Example 4] Tryptophan 3 mM and isatin 0.12 mM

[0150] It was manufactured in the same manner as Comparative Example 2, but instead of adding 10 mM tryptophan, it was manufactured by adding 3 mM tryptophan and 0.12 mM isatin.

[0151] Here, the above isatin is produced by decomposing 0.08 mM indigo extracted from jeans with UV at 254 nm.

[0152] [Example 5] Isatin 0.12 mM

[0153] It was manufactured in the same manner as Comparative Example 2, but instead of adding 10 mM tryptophan, it was manufactured by adding only 0.12 mM isatin.

[0154] Here, the above isatin is produced by decomposing 0.08 mM indigo extracted from jeans with UV at 254 nm.

[0155] [Example 6] Isatin 0.04 mM

[0156] It was manufactured in the same manner as Comparative Example 2, but instead of adding 10 mM tryptophan, it was manufactured by adding only 0.04 mM isatin.

[0157] Here, the above isatin is produced by decomposing indigo extracted from jeans with UV at 254 nm.

[0158]

[0159] [Experimental Example 1] Comparison of indirubin and indigo contents according to isatin content when manufactured by adding Tween 80

[0160] Figure 3 shows the results of comparing the amount of indirubin and indigo produced when indirubin was manufactured by adding Tween 80 according to one experimental example of the present invention.

[0161] Referring to Figure 3, when 7% of Tween 80 was added, it was confirmed that the content of indirubin increased and the content of indigo decreased depending on the ratio of isatin and tryptophan.

[0162] [Experimental Example 2] Comparison of indirubin and indigo contents according to isatin content when manufactured without adding Tween 80.

[0163] Figure 4 shows the results of comparing the production amounts of indirubin and indigo when indirubin is manufactured without adding Tween 80 according to an experimental example of the present invention.

[0164] Referring to Fig. 4, it was confirmed that the content of indirubin increased and the content of indigo decreased as the amount of isatin increased even when Tween 80 was not added. Specifically, when the amount of tryptophan was fixed at 3 mM and the amount of isatin was compared from 1 mM to 6 mM, it was confirmed that the largest amount of indirubin was produced when isatin was 2 mM.

[0165] [Experimental Example 3] Comparison of indirubin production according to tryptophan content when isatin concentration is fixed.

[0166] Figure 5 shows the results of comparing the amount of indirubin produced according to the tryptophan content when the concentration of isatin is fixed according to one experimental example of the present invention.

[0167] Referring to Figure 5, when satin was fixed at 2 mM, the amount of indirubin produced was compared according to the amount of tryptophan, and it was confirmed that the production efficiency was the best when tryptophan was 2 mM, but the amount produced itself was the highest when tryptophan was 3 mM.

[0168] [Experimental Example 4] Confirmation of indirubin production by adding isatin formed by decomposing synthetic indigo under UV (254 nm) to the culture medium.

[0169] Figure 6 shows the results of measuring the amount of indirubin produced by adding isatin, which is formed by decomposing synthetic indigo with UV (254 nm) according to an experimental example of the present invention, to a culture medium.

[0170] Referring to Fig. 6, when 2 mM of synthetic indigo was decomposed by UV, it was confirmed that 3 mM of isatin was produced, and when 2 mL of this was added to the culture medium and reacted, it was confirmed that more indirubin was produced than when only tryptophan was added as a substrate or when no substrate was added. In addition, when indigo was produced with tryptophan present in the LB medium without adding a separate substrate such as tryptophan (lower graph of Fig. 6), it was confirmed that indirubin was produced when isatin was added.

[0171] [Experimental Example 5] Production of indirubin was confirmed by adding isatin formed by decomposing indigo extracted from jeans under UV (254 nm) to the culture medium.

[0172] Figure 7 shows the results of measuring the amount of indirubin produced by adding isatin, which is formed by decomposing indigo extracted from jeans under UV (254 nm), to a culture medium.

[0173] Referring to Figure 7, when indigo was extracted from jeans with DMSO and decomposed with UV, it was confirmed that 1 mM isatin was produced, and when 2 mL of this was added to the culture medium and reacted, it was confirmed that more indirubin was produced than when no substrate was added.

[0174]

[0175] 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.

[0176] 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 solution 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 adding isatin to the primary culture medium; 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 isatin is converted into indirubin by the above E. coli and the above enzyme, Method for manufacturing indirubin.

2. In paragraph 1, In the step of forming the secondary culture medium, a substrate is additionally mixed into the primary culture medium. Method for manufacturing indirubin.

3. In paragraph 2, The above substrate comprises L-tryptophan or indole, Method for manufacturing indirubin.

4. In paragraph 3, The substrate is converted into indirubin by the above E. coli and the above enzyme, Method for manufacturing indirubin, 5. In paragraph 1, The above isatin is produced by decomposing indigo with UV. Method for manufacturing indirubin.

6. In paragraph 5, The above indigo is extracted from jeans. Method for manufacturing indirubin.

7. 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 multiplying the E. coli and the enzyme.

8. 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).

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

10. In paragraph 9, A method for producing indirubin, wherein the above E. coli contains tryptophanase (tnaA), which is an endogenous E. coli gene.

11. In paragraph 1, The above primary culturing step is, 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.

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

13. In paragraph 12, 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.

14. In paragraph 13, A method for producing indirubin, wherein the concentration of the surfactant in the secondary culture medium is 0.1% to 10%.

15. In paragraph 12, 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.

16. In paragraph 12, A method for producing indirubin, wherein the sulfur-containing material comprises a substance selected from the group consisting of FeSO4, Na2SO4, (NH4)2SO4, MgSO4, methionine, cysteine, and combinations thereof.

17. In paragraph 1, A method for producing indirubin, wherein the above tertiary culture medium contains indigo and indirubin.

18. In paragraph 17, A method for producing indirubin, wherein the above solvent selectively extracts indirubin.

19. 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.

20. Indirubin, manufactured by the method according to any one of claims 1 to 19.

21. A pharmaceutical composition comprising indirubin according to Article 20.

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