Novel water-soluble melanin and method for preparing same

Abstract

The present invention relates to a novel water-soluble melanin and a method for preparing same. The method for preparing a water-soluble melanin of the present invention can produce a significantly large amount of melanin compared to an existing melanin production method, and, thus, provides good productivity for using a water-soluble melanin commercially. In addition, the water-soluble melanin of the present invention is dissolved in water rather than an organic solvent and can, thus, overcome existing limitations, and has an excellent antioxidant ability, growth inhibitory ability against fungi, and whitening effect compared to other existing synthetic melanins.

Description

Novel water-soluble melanin and method for producing the same

[0001] The present invention relates to a novel water-soluble melanin and a method for producing the same.

[0002] Melanin is a general term for high-molecular compounds formed by oxidation and polymerization of phenol or indole compounds. Its brown or black color is characteristic and used as a pigment. Melanin is widely distributed throughout the plant and animal kingdoms, and its pigment determines the color of skin and hair in humans and animals. Because melanin is insoluble, amorphous, and has a variable shape, it is currently difficult to accurately analyze its structure. Furthermore, melanin is insoluble in water and tends to dissolve primarily in organic solvents, limiting its applications.

[0003] Research is being conducted to increase the efficiency of melanin synthesis by controlling the expression or activity levels of tyrosinase, a key enzyme in the melanin synthesis pathway, and its related proteins. The present inventors, through their prior application, Republic of Korea Patent Application Publication No. 10-2020-0140636 (December 16, 2020), disclosed a method for biosynthesizing a novel indigo-based polymer, i.e., a melanin-like polymer based on an indigoid-based monomer (aka melanindigo), using tyrosine as a substrate in E. coli overexpressing CYP102G4 and melC.

[0004] Meanwhile, gallic acid (3,4,5-trihydroxybenzoic acid) is a major component of tannin, which is found in large quantities in green tea, black tea, wine, coffee, and cornelian cherry. It is also known to be abundant in food products as well as herbal medicines such as Cornus officinalis, Schisandra chinensis, Hawthorn berry, and Galla chinensis. Gallic acid is well known for its physiochemical properties such as scavenging active oxygen, repairing damaged DNA, neuroprotective effects, gastrointestinal protection, and anticancer effects.

[0005] To date, no method has been known to produce water-soluble melanin, unlike conventional insoluble melanin compounds, using gallic acid as a substrate in E. coli expressing the melC gene.

[0006] The present invention aims to provide a method for producing water-soluble melanin, which comprises a step of culturing a recombinant E. coli expressing a melC gene using gallic acid as a substrate.

[0007] In addition, the present invention aims to provide a novel water-soluble melanin that is soluble in water and produced by the above production method.

[0008] In order to solve the above problem, the inventors of the present invention, while studying a biotechnology method for more effectively producing melanin, cultured E. coli expressing the melC gene using gallic acid as a substrate, and as a result, were able to produce a significantly larger amount of melanin than the existing melanin production method, and also confirmed that the obtained melanin compound is highly soluble in water and has excellent antioxidant activity, fungal growth inhibition activity, and whitening effect compared to other existing synthetic melanins, thereby completing the present invention.

[0009] Accordingly, the present invention provides a method for producing water-soluble melanin, which comprises a step of culturing a recombinant E. coli expressing a melC gene using gallic acid as a substrate.

[0010] In one embodiment, the melC gene may encode tyrosinase.

[0011] In one embodiment, the melC gene may be derived from Bacillus megaterium.

[0012] In one embodiment, the recombinant E. coli expressing the melC gene may be a recombinant strain prepared by introducing the melC gene into Escherichia coli BL21(DE3), Escherichia coli BL21(DE3)::melC.

[0013] In one embodiment, the gallic acid may be added at a concentration of 3 mM or more.

[0014] In one embodiment, the gallic acid may be added at a concentration of 20 mM or more.

[0015] In one embodiment, the manufacturing method may further comprise a step of recovering water-soluble melanin from a culture medium of E. coli.

[0016] In one embodiment, the water-soluble melanin may include a compound represented by the following chemical formula 1:

[0017] [Chemical Formula 1]

[0018]

[0019] In the above formula, n is an integer greater than or equal to 1.

[0020] The present invention also provides a water-soluble melanin produced by the above production method.

[0021] In one embodiment, the water-soluble melanin may include a compound represented by the following chemical formula 1:

[0022] [Chemical Formula 1]

[0023]

[0024] In the above formula, n is an integer greater than or equal to 1.

[0025] The present invention also provides a water-soluble melanin produced by the above production method.

[0026] In one embodiment, the water-soluble melanin may include a compound represented by the following chemical formula 1:

[0027] [Chemical Formula 1]

[0028]

[0029] In the above formula, n is an integer greater than or equal to 1.

[0030] The present invention also provides a whitening cosmetic composition comprising the water-soluble melanin as an additive.

[0031] The method for producing water-soluble melanin, which includes a step of culturing recombinant E. coli expressing the melC gene using the gallic acid of the present invention as a substrate, can produce a significantly larger amount of melanin than conventional melanin production methods, and thus has excellent productivity for commercial use of water-soluble melanin.

[0032] In addition, existing melanins tend to be insoluble in water and mainly soluble in organic solvents, which limits the application of melanin. However, the water-soluble melanin of the present invention can overcome existing limitations by soluble in water rather than organic solvents, and has superior antioxidant activity, fungal growth inhibition, and whitening effects compared to other existing synthetic melanins.

[0033] Figure 1 schematically illustrates the method of the present invention for biosynthesizing water-soluble gallic acid melanin using gallic acid as a substrate in E. coli expressing the melC gene.

[0034] Figure 2 shows the production amount of water-soluble gallic acid melanin according to the amount of gallic acid added.

[0035] Figure 3 shows the water-soluble gallic acid melanin of the present invention dissolved in water.

[0036] Figure 4 shows the results of FE-SEM-EDS analysis of the water-soluble gallic acid melanin of the present invention.

[0037] Figure 5 shows the FTIR spectrum results of the water-soluble gallic acid melanin of the present invention.

[0038] Figure 6 shows a scanning electron microscope image of the water-soluble gallic acid melanin of the present invention.

[0039] Figure 7 shows the results of an antibacterial test of the water-soluble gallic acid melanin of the present invention against gram-negative and gram-positive bacteria.

[0040] Figure 8 shows the results of an antioxidant activity test of the water-soluble gallic acid melanin of the present invention.

[0041] Figure 9 shows the results of a whitening effect test of the water-soluble gallic acid melanin of the present invention.

[0042] Hereinafter, the present invention will be described in detail.

[0043] In one aspect, the present invention provides a method for producing water-soluble melanin, comprising the step of culturing a recombinant E. coli expressing a melC gene using gallic acid as a substrate.

[0044] The melC gene was previously described in the literature Regioselective Hydroxylation of trans-Resveratrol via Inhibition of Tyrosinase from Streptomyces avermitilis MA4680, N Lee, EJ Kim, BG Kim - ACS chemical biology, 2012 - ACS Publications. In the present invention, the melC gene information described in the literature was used as is.

[0045] In the manufacturing method of the present invention, the melC gene may encode tyrosinase. More specifically, the melC gene may be derived from Bacillus megaterium, but is not limited thereto.

[0046] In the manufacturing method of the present invention, the recombinant E. coli expressing the melC gene may be a recombinant strain, Escherichia coli BL21(DE3)::melC, prepared by introducing the melC gene into Escherichia coli BL21(DE3). The strain may be prepared without limitation using genetic information and / or genetic recombination methods known in the art.

[0047] In the production method of the present invention, the gallic acid used as a substrate may be added to the culture medium at a concentration of 3 mM or more, preferably at a concentration of 5 mM or more, more preferably at a concentration of 10 mM or more, even more preferably at a concentration of 15 mM or more, and most preferably at a concentration of 20 mM or more. However, the amount of gallic acid is not limited thereto, and may be added at a concentration within a range expected to enable efficient melanin synthesis by the recombinant E. coli.

[0048] In the manufacturing method of the present invention, the cultivation of recombinant E. coli expressing the melC gene can be performed using a culture medium based on LB medium (Luria-Bertani broth). Specifically, the culture medium may additionally contain, but is not limited to, CuSO4 and 5-aminolevulinic acid (ALA), which are coenzymes necessary for melC expression. In addition, the culture medium may additionally contain, but is not limited to, tryptone, yeast extract, and sodium chloride.

[0049] In the manufacturing method of the present invention, the cultivation of the recombinant E. coli expressing the melC gene is preferably performed at a temperature of 20 to 40°C for 15 to 40 hours, and specifically, it is more preferably performed at a temperature of 30 to 37°C for 20 to 30 hours, but is not limited thereto. In one embodiment, the cultivation of the E. coli may be performed at 37°C, and the expression of melC may be achieved at 30°C, but is not limited thereto, and may be appropriately adjusted by a person skilled in the art in consideration of conditions suitable for general strain cultivation and / or conditions required for enzyme expression. In addition, the cultivation may be performed at a cycle of 200 rpm, but is not limited thereto.

[0050] In the manufacturing method of the present invention, a step of recovering water-soluble melanin from the culture solution of the E. coli may be additionally included after the step of culturing a recombinant E. coli expressing the melC gene using gallic acid as a substrate. Specifically, the recovery step includes, but is not limited to, the steps of centrifuging the culture solution, collecting only the supernatant, adjusting the pH to 2, centrifuging the settled melanin again, and washing and drying.

[0051] As shown in the examples described below, when 20 mM gallic acid was added to 1 L of the medium according to the manufacturing method of the present invention, about 2.8 g / L of melanin was produced. When the same melC gene is expressed and tyrosine is used as a substrate, the amount of melanin produced is 120 mg / L (see Microbial Production of Melanin Pigments from Caffeic Acid and L-Tyrosine Using Streptomyces glaucescens and FCS-ECH-Expressing Escherichia coli, Soo-Yeon Ahn, Seyoung Jang, Pamidimarri DVN Sudheer and Kwon-Young Choi), so the amount of about 2.8 g / L produced is quite large. Therefore, the manufacturing method of the present invention can produce a significantly larger amount of melanin than the existing melanin production method, and has good productivity for commercial use of water-soluble melanin.

[0052] In another aspect, the present invention provides a water-soluble melanin produced by the above production method.

[0053] The term "water-soluble melanin" as used herein may be used interchangeably with "gallic acid melanin", "water-soluble gallic acid melanin", "gallic acid-based water-soluble melanin", and "water-soluble melanin compound".

[0054] The water-soluble melanin of the present invention may include a compound represented by the following chemical formula 1:

[0055] [Chemical Formula 1]

[0056]

[0057] In the above formula, n is an integer greater than or equal to 1.

[0058] Specifically, in the compound represented by the above chemical formula 1, the value of n is an integer greater than or equal to 1, and more specifically, may be in the range of 1 to 10,000, in the range of 1 to 5,000, or in the range of 2 to 1,000, but is not limited thereto.

[0059] Conventional melanin tends to be insoluble in water and mainly soluble in organic solvents, which limits the application of melanin. However, the water-soluble melanin of the present invention can overcome the existing limitations by dissolving in water rather than in organic solvents, and has superior antioxidant activity, fungal growth inhibition, and whitening effects compared to other conventional synthetic melanins, so it can be used as a cosmetic additive.

[0060] Therefore, another aspect of the present invention provides a whitening cosmetic composition comprising the water-soluble melanin as an additive.

[0061] The above cosmetic composition may include a cosmetically effective amount of the water-soluble melanin and a cosmetically acceptable carrier, wherein the cosmetically effective amount means an amount sufficient to achieve a whitening effect.

[0062] The cosmetic composition of the present invention may contain commonly used auxiliary agents such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, fragrances, fillers, blocking agents, pigments, deodorants, and dyes.

[0063] In addition, it is expected that the water-soluble melanin of the present invention can increase electrical conductivity by coating it on an insulator or a conductor, and therefore, a material coated with the water-soluble melanin of the present invention can be applied as a sensor or capacitor.

[0064] Hereinafter, the composition and effects of the present invention will be described in more detail through examples. However, these examples are intended only to exemplify the present invention, and the scope of the present invention is not limited by these examples.

[0065]

[0066] Example

[0067] Example 1: Biosynthesis of water-soluble gallic acid melanin using gallic acid as a substrate.

[0068] Escherichia coli strain BL21 (DE3) was subcultured and cultured until the absorbance reached 0.6 to 0.7 at a wavelength of 600 nm. After strain BL21 (DE3) was grown to an absorbance of 0.6 to 0.7, 0.25 mM IPTG (isopropylthio-β-galactoside) was added to induce IPTG to express the melC gene (derived from Bacillus megaterium). Gallic acid, a substrate, was added at 5 to 20 mM along with gene expression to compare the final melanin production. After 24 hours of gene expression, the culture medium was collected and centrifuged at 8000 rpm, 4℃, for 10 minutes. To isolate melanin, only the supernatant obtained by centrifugation was collected and the pH was adjusted to 2 with hydrochloric acid. The supernatant, whose pH was adjusted to 2, was allowed to stand for 4 hours, and the settled melanin was obtained by centrifugation (8000 rpm, 4℃, 10 minutes). The melanin thus obtained was washed three times with distilled water and dried in an oven at 60℃ for one day to obtain the final product. Each experiment was repeated three times.

[0069] The results of melanin production according to the amount of gallic acid added as a substrate are shown in Fig. 2. As shown in Fig. 2, when 20 mM gallic acid was added per 1 L of medium, approximately 2.8 g / L of melanin was produced (see Fig. 2). When the same melC gene was expressed and tyrosine was used as a substrate, the amount of melanin produced was 120 mg / L (see the literature, Microbial Production of Melanin Pigments from Caffeic Acid and L-Tyrosine Using Streptomyces glaucescens and FCS-ECH-Expressing Escherichia coli, Soo-Yeon Ahn, Seyoung Jang, Pamidimarri DVN Sudheer and Kwon-Young Choi), so it can be seen that the production amount of approximately 2.8 g / L is a considerably large amount.

[0070] Meanwhile, the obtained melanin exhibits a property of being highly soluble in water, unlike conventional melanin. The water-soluble gallic acid melanin of the present invention dissolved in water is shown in Figure 3.

[0071] From the above, it was determined that the biosynthetic method of the present invention using gallic acid as a substrate in E. coli expressing the melC gene has good productivity for commercial use of water-soluble melanin.

[0072]

[0073] Example 2: Molecular Structure Analysis of Water-Soluble Gallic Acid Melanin

[0074] In order to predict the molecular structure of the water-soluble gallic acid melanin obtained in Example 1, the elemental content was measured using energy dispersive spectroscopy. Specifically, field emission scanning electron microscopy (FE-SEM-EDS) was analyzed using a JSM-7900F model from JEOL, and Fourier transform infrared (FTIR) was analyzed using a Nicolet iS50 model from Thermo.

[0075] The results of FE-SEM-EDS analysis of water-soluble gallic acid melanin and the measured ratios of carbon, nitrogen, and oxygen are shown in Fig. 4. In addition, the results of FTIR spectrum are shown in Fig. 5. Referring to the measured ratios of carbon, nitrogen, and oxygen and the FTIR molecular peaks of Fig. 5, the molecular structure of water-soluble gallic acid melanin was predicted as shown in the following chemical formula.

[0076] [Chemical Formula 1]

[0077]

[0078] In the above formula, n is an integer greater than or equal to 1.

[0079] The porosity of the surface of each sample of a number of melanins, including the water-soluble gallic acid melanin obtained in Example 1, was observed at magnifications of 10x, 100x, 500x, 1000x, 5000x, and 10000x using a field emission scanning electron microscope (FE-SEM) (JEOL JSM-7900F model).

[0080] As shown in Fig. 6, the water-soluble gallic acid melanin of the present invention was observed to have a very smooth surface even under high magnification, unlike conventional melanins. Therefore, the water-soluble gallic acid melanin of the present invention is highly likely to be smoothly adsorbed onto the surface of a material to be applied rather than forming porosity when coated or sprayed onto the material, and thus, it is expected that the physiological, physical, and electrical properties of water-soluble melanin can be uniformly obtained.

[0081]

[0082] Example 3: Antibacterial test of water-soluble gallic acid melanin

[0083] The antibacterial activity of the water-soluble gallic acid melanin obtained in Example 1 against gram-negative bacteria (E. coli) and gram-positive bacteria (C. albican) was tested. Specifically, the antibacterial activity test was performed on gram-negative bacteria (E. coli) and gram-positive bacteria (C. albican), respectively, and the effect when soluble gallic acid melanin was present at a concentration of 1 mg / ml in a culture medium was measured. Each strain was subcultured, and after 24 hours, the absorbance at 600 nm was measured to determine the bacterial growth inhibition ability of the water-soluble gallic acid melanin. Each experiment was repeated three times.

[0084] As a result, as shown in Fig. 7, compared to the control group without melanin, it exhibited an inhibitory effect of about 4.6% against E. coli and an inhibitory effect of 25.7% against Candida albicans.

[0085] In this way, it was determined that the water-soluble gallic acid melanin of the present invention can be applied to reduce infectivity by showing better growth inhibition ability against fungi (Candida).

[0086]

[0087] Example 4: Antioxidant activity test of water-soluble gallic acid melanin

[0088] The antioxidant activity of the water-soluble gallic acid melanin obtained in Example 1 was tested. Specifically, the antioxidant test was conducted using ABTS (2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) radical scavenging activity. ABTS was dissolved in phosphate-buffered saline (PBS) solution to make 14.8 mM. A 4.9 mM potassium persulfate solution was prepared, mixed 1:1 with the previously prepared 14.8 mM ABTS solution, and allowed to stand at room temperature for 22-23 hours. A working solution was prepared such that the absorbance at 734 nm was 0.7±0.05. The prepared working solution was mixed with 20 μl of the sample and left in the dark for 10 minutes. The absorbance of the leftover sample was measured at 734 nm, and the antioxidant capacity was evaluated using the following formula:

[0089] AA% = [Background(ABTS)-S(sample+ABTS)] / Background(ABTS)Х100.

[0090] Each experiment was repeated three times.

[0091] The results of measuring the antioxidant capacity of water-soluble gallic acid melanin are shown in Fig. 8. As shown in Fig. 8, the IC of water-soluble gallic acid melanin 50 The value is approximately 66±2.09 μg / ml, showing significantly better antioxidant activity compared to other melanins (see Table 1 below).

[0092] Table 1: Antioxidant effects of melanin

[0093]

[0094] As such, it was determined that the water-soluble gallic acid melanin of the present invention has excellent antioxidant activity and thus would be advantageous for application as a cosmetic additive.

[0095]

[0096] Example 5: Whitening effect test of water-soluble gallic acid melanin

[0097] To test the whitening effect of the water-soluble gallic acid melanin obtained in Example 1, a tyrosinase inhibition assay was performed. Specifically, the whitening test used Biovision's tyrosinase inhibition screening. After dissolving the inhibitor in an appropriate solvent, the sample was diluted to a concentration five times the desired test concentration using tyrosinase assay buffer. An inhibition control (kojic acid) was used as a control. Next, 20 μL of the diluted inhibitor was added to a 96-well plate. Then, 50 μL of tyrosinase enzyme solution (48 μL of tyrosinase assay buffer, 2 μL of tyrosinase) was added to each well, mixed well, and incubated at 25°C for 10 minutes. Then, 30 μL of tyrosinase substrate solution (23 μL of tyrosinase assay buffer, 2 μL of tyrosinase substrate, and 5 μL of tyrosinase enhancer) was added to each well. The absorbance at 510 nm was then monitored for 30–60 min in kinetic mode, and two time points within the linear range of the plot were selected to calculate the inhibitory activity. The slope (calculated by dividing net ΔAbs(Abs2-Abs1) by time ΔT(T2-T1)) was determined for all samples, including the enzyme activity control (EC). The inhibitory activity (%) of each sample was calculated according to the following formula:

[0098] Relative inhibition (%) = (slope EC - slope sample) / slope ECХ100 (%)

[0099] The results are shown in Fig. 9. As shown in Fig. 9, the water-soluble gallic acid melanin of the present invention exhibited a whitening effect of about 37% at a concentration of 0.0625 mg / ml, and the whitening effect was better at 0.0625 mg / ml than at 0.125 mg / ml due to the influence of the color of melanin. Through this, it was determined that the water-soluble gallic acid melanin of the present invention can be expected to exhibit a whitening effect when applied as a cosmetic additive.

Claims

1. A method for producing water-soluble melanin, comprising the step of culturing recombinant E. coli expressing the melC gene using gallic acid as a substrate.

2. A manufacturing method according to claim 1, wherein the melC gene encodes tyrosinase.

3. A manufacturing method according to claim 1, wherein the melC gene is derived from Bacillus megaterium.

4. A manufacturing method according to claim 1, wherein the recombinant E. coli expressing the melC gene is a recombinant strain prepared by introducing the melC gene into Escherichia coli BL21(DE3), Escherichia coli BL21(DE3)::melC.

5. A manufacturing method in claim 1, wherein gallic acid is added at a concentration of 3 mM or more.

6. A manufacturing method in claim 1, wherein gallic acid is added at a concentration of 20 mM or more.

7. A manufacturing method according to claim 1, further comprising a step of recovering water-soluble melanin from a culture solution of E. coli.

8. A manufacturing method according to claim 1, wherein the water-soluble melanin comprises a compound represented by the following chemical formula 1: [Chemical Formula 1] In the above formula, n is an integer greater than or equal to 1.

9. Water-soluble melanin manufactured by a manufacturing method according to any one of claims 1 to 8.

10. In claim 9, a water-soluble melanin comprising a compound represented by the following chemical formula 1: [Chemical Formula 1] In the above formula, n is an integer greater than or equal to 1.

11. A whitening cosmetic composition containing the water-soluble melanin of Article 9 as an additive.

12. A whitening cosmetic composition containing the water-soluble melanin of Article 10 as an additive.

Images