KR102278598B1 - Novel indigoid polymer and biosynthesis thereof - Google Patents

Abstract

The present invention relates to novel indigo-based polymers, biosynthetic methods and uses thereof.

Description

Novel indigo-based polymer and biosynthesis method thereof

The present invention relates to novel indigo-based polymers, biosynthetic methods and uses thereof.

Dyes are pigments that dye threads, fabrics, and fabrics, and are distinguished from pigments that do not dissolve in water, oil, or alcohol. The dyes are divided into natural dyes and synthetic dyes according to their origin, and in the case of natural dyes, the bond with fibers is weak and color development is not good, so it is dyed with a mordant such as alum.

Before chemistry developed like today, ferric oxide (Fe ₂ O ₃ ) was used for coloring, or the pigment of insects or plants, such as cochineal, a type of shellfish, was extracted and used as a dye. Natural dyes are divided into animal dyes, vegetable dyes, mineral dyes, etc., depending on the method of collecting the dyes obtained from nature. It was from the mid-19th century that various colors could be produced by synthesizing colored organic substances (dyes) without relying on natural pigments. This era was the era of so-called modern industry, where acid/alkali industries and coke-based ironmaking took place. In England, coal was reconstituted and the supply of city gas began.

In the process of synthesizing quinine from toluidine, which is a component of coal tar, while carbonizing coke for iron manufacture and conducting research on coal tar, which is the remaining residue, we accidentally discovered a brightly colored pigment, which made silk beautiful. It can be colored purple, hence the name mauvein. After that, a synthetic dye company called Mobra was established by improving this dye. Furthermore, with the progress of research on the structure and synthesis of aromatic compounds centered on benzene, basic theories on color development such as chromophores and auxochromes have emerged. The synthesis of dyes has been gradually developed thanks to such basic chemical research, development of synthesis technology, and changes in industrial form, and the most important event among them is the synthesis of alizarin and indigo.

Alizarin is a pigment extracted from the roots of western marigolds, and has been used for red dyeing cotton and the like since ancient times. Later, he discovered that alizarin was an anthracene derivative and succeeded in synthesizing it from anthracene in 1868. This made it possible to mass-produce alizarin without relying on natural raw materials.

Indigo is a pigment extracted from indigo, and it has been used in Egypt since about 2,000 to 1,000 BC because it is well dyed with animal fibers such as wool or silk yarn or vegetable fibers such as cotton and hemp. Indigo is dyed a beautiful indigo blue and is a dye with good weather resistance. Indigo, the raw material, was cultivated in large quantities mainly in India, and was synthesized by a German buyer in 1880. Since the synthesis method was improved and simple synthesis became possible, the use of natural indigo was significantly reduced, and the cultivation of indigo rapidly declined.

The synthesis of these two dyes was a dramatic shift from the conventional method that relied on nature to the method of chemical synthesis. Since then, the path of chemical synthesis has been rapidly expanded, and further, as a result of research on synthetic dyes centered on the tar industry in the mid-19th century, tens of thousands of synthetic dyes have been developed and the foundation of the synthetic pharmaceutical industry has been established. did it

The main use of these dyes is dyeing textiles, but today, the range of use has expanded from leather, fur, paper, edible oils and fats to fuels. It is also widely used in various inks, photosensitive pigments, and medicine. For such a wide range of uses, about 40,000 types of synthetic dyes and 2,500 items are currently made, and they are used according to each purpose.

As a result of intensive research efforts to provide a novel dye material without toxicity, the present inventors have used a strain overexpressing a specific enzyme, such as a melC and CYP102G4 overexpressing strain, from L-tyrosine to melanin based on an indigoid-based monomer. By synthesizing a similar polymer, aka melanindigo, it was confirmed that it exhibits superior dyeing power and/or color development power compared to conventional biosynthesized melanin or indigo, and completed the present invention.

As one aspect for achieving the above object, the present invention provides a compound represented by the following formula (1):

[Formula 1]

In Formula 1,

는 단일결합 또는 이중결합으로 이에 관여하는 탄소의 결합가(valence)를 충족시키도록 결정되며,

is determined to satisfy the valence of the carbon involved in it with a single bond or a double bond,

R ₁ to R ₄ are each independently hydrogen or absent,

R ₁ and R ₂ and R ₃ and R ₄ are the same as each other,

는 이중결합이고,connected to that O

is a double bond,

Polymerized with a neighboring monomer through one or more sites of L ₁ to L _4,

n is an integer from 2 to 10,000.

For example, the polymer represented by Formula 1 may be a polymer formed by homogeneous or heterogeneous polymerization of one or more monomers selected from compounds represented by Formulas 2 to 4:

[Formula 2]

[Formula 3]

[Formula 4]

.

.

For example, the compound of the present invention may be prepared by biosynthesis from tyrosine using melC and CYP102G4, but is not limited thereto. Specifically, L-tyrosine is 3,5,6-trihydroxyindole (3,5,6-trihydroxyindole; THI, IUPAC name 1H-indole-3,5 as an intermediate product by a series of reactions by melC and CYP102G4). ,6-triol; 1H-indole-3,5,6-triol), indoxyl-5,6-quinone (IUPAC name 3-hydroxy-1H-indole-5,6 -dione; 3-hydroxy-1H-indole-5,6-dione), and three kinds of monomers represented by the above formulas 2 to 4, 2-(5,6- Dioxo-3-oxo-1H-indol-2-ylidene)-5,6-dioxo-1H-indol-3-one (2-(5,6-dioxo-3-oxo-1H-indol-2) -ylidene)-5,6-dioxo-1H-indol-3-on), 2-(5,6-dihydroxy-3-oxo-1H-indol-2-ylidene)-5,6-dioxo -1H-indol-3-one (2-(5,6-dihydroxy-3-oxo-1H-indol-2-ylidene)-5,6-dioxo-1H-indol-3-on), 2-(5 ,6-dihydroxy-3-oxo-1H-indol-2-ylidene)-5,6-dihydroxy-1H-indol-3-one (2-(5,6-dihydroxy-3-oxo- 1H-indol-2-ylidene)-5,6-dihydroxy-1H-indol-3-on) or mixtures thereof. Furthermore, these compounds can be used as monomers in the subsequent polymerization reaction.

In another aspect, the present invention provides a method for preparing a compound represented by the following formula (1), comprising culturing a strain overexpressing CYP102G4 and melC while providing tyrosine:

[Formula 1]

In Formula 1,

는 단일결합 또는 이중결합으로 이에 관여하는 탄소의 결합가(valence)를 충족시키도록 결정되며,

is determined to satisfy the valence of the carbon involved in it with a single bond or a double bond,

R ₁ to R ₄ are each independently hydrogen or absent,

R ₁ and R ₂ and R ₃ and R ₄ are the same as each other,

는 이중결합이고,connected to that O

is a double bond,

It is polymerized with a neighboring monomer through one or more sites of L ₁ to L ₄ , and hydrogen is bonded to the remaining sites that do not participate in polymerization,

n is an integer from 2 to 10,000.

For example, the strain overexpressing CYP102G4 and melC used in the production method of the present invention is Escherichia Recombinant strain Escherichia prepared by introducing foreign genes for melC and CYP102G4 expression into coli BL21(DE3) coli BL21(DE3):: melC :: cyp102G4 . The strain can be prepared using, without limitation, genetic information and/or genetic recombination methods known in the art.

For example, the culture of the strain can be performed using a culture medium based on LB medium (luria-bertani broth).

Specifically, the culture medium may additionally contain a heme precursor, which is a precursor necessary for CYP102G4 protein expression, CuSO ₄ and 5-aminolevulinic acid (ALA), which are coenzymes necessary for melC expression. , but not limited thereto.

Furthermore, the culture medium may further include tryptone, yeast extract, and sodium chloride, but is not limited thereto.

For example, the culture of the strain may be carried out at 37 ° C., and the overexpression of enzymes, that is, CYP102G4 and melC, may be achieved at 30 ° C., but is not limited thereto, and conditions and / or enzymes suitable for conventional strain culture. A person skilled in the art can appropriately control it in consideration of the conditions required for overexpression. In addition, the culture may be aerated at a cycle of 200 rpm, but is not limited thereto.

As another aspect, the present invention provides a dye composition comprising the compound represented by Formula 1 above.

Since the dye composition of the present invention contains melanindigo biosynthesized from the amino acid tyrosine as an active ingredient, it may be non-toxic and not harmful to the human body.

The dye composition of the present invention may be used for dyeing paper, fiber or fabric, but is not limited thereto. Furthermore, these dyed papers, fibers, and fabrics are mainly used for packaging materials, clothes, and ornaments, and the fact that they are not toxic to the human body is one of the important factors that a dye composition used for this should have.

The dye composition of the present invention may further include additives such as solubilizers, buffers, and anti-freezing agents. The solubilizer is an additive component that prevents aggregation of the dye and helps dissolve, and typical examples include urea, thiourea, caprolactam, and the like. The buffer is a component that serves to buffer the pH in order to prevent the decomposition of the dye during storage and storage, and representative examples include sodium phosphate, potassium phosphate, sodium acetate, and the like. The anti-freeze agent is a component that lowers the freezing point of the liquid and prevents the liquid dye from freezing during storage in a liquid state, and typical examples include ethylene glycol, polyethylene glycol, polyhydric alcohol compounds, and the like. In this case, the additive may be added in an amount of about 1 to 20% by weight based on the total weight of the composition, but is not limited thereto.

Since the novel indigo-based polymer of the present invention is a material synthesized from tyrosine, an amino acid by biosynthesis by enzyme action, it does not show toxicity to the human body, and has excellent color development and / or coloring power compared to conventional indigo or melanin, so it is useful as a dye composition can be used

1 is a diagram showing the chemical composition of tyrosine melanin according to Comparative Example 1 and 15T FT-ICR mass spectrometry of melanindigo of the present invention.
2 is a diagram showing the molecular distribution of tyrosine melanin according to Comparative Example 1 and melanin-digo of the present invention.
3 is a diagram showing the color difference of indigo synthesized by CYP102G4 overexpressing E. coli, melanin synthesized by melC overexpressing E. coli, and melanindigo synthesized by melC and CYP102G4 overexpressing E. coli with the naked eye.
4 is a (left) SEM image of melanin synthesized by melC overexpressing E. coli, and melanindigo synthesized by melC and CYP102G4 overexpressing E. coli, and (right) a diagram showing the degree of color development during cellulose staining.
5 is a diagram showing the RGB extraction results of melanin synthesized by melC overexpressing E. coli, and melanindigo synthesized by melC and CYP102G4 overexpressing E. coli.

Hereinafter, the present invention will be described in more detail through examples. These Examples are for explaining the present invention in more detail, and the scope of the present invention is not limited by these Examples.

Example One: melC and CYP102G4 L- using overexpressed E. coli from tyrosine Synthesis of indigoid-based melanin

[Scheme 1]

Escherichia , a recombinant strain prepared by introducing foreign genes for melC and CYP102G4 expression into melC and CYP102G4 overexpressing strains, such as Escherichia coli BL21 (DE3) while supplying L-tyrosine, Escherichia coli BL21(DE3):: melC :: cyp102G4 was cultured to biosynthesize indigo. A specific synthesis mechanism is shown in Scheme 1 above. Specifically, the indole derivative obtained as an intermediate by CYP102G4 is dimerized using a hydroxy group introduced on the pyrrole ring to form a compound including an indigoid structure, and a catechol structure formed by melC By polymerization through, an indigo polymer, a final product, was synthesized, which was named melanindigo.

Specifically, as a melanindigo biosynthetic strain, Escherichia to prepare a melC and CYP102G4 introducing a foreign gene for expression prepared by the recombinant strain, Escherichia coli BL21 (DE3) :: melC :: cyp102G4 on coli BL21 (DE3), based on LB medium (luria-bertani broth) CYP102G4 It was cultured in a culture medium containing a heme precursor, a precursor required for protein expression, 5-aminolevulinic acid (ALA), CuSO _{4, a coenzyme required for melC expression, and L-tyrosine as a substrate.} Specifically, the growth medium was prepared by adding 10.0 g of tryptone, 5.0 g of yeast extract and 10.0 g of sodium chloride to 1 L of LB medium. The strain was cultured at 37°C using the culture medium, but overexpression of melC and CYP102G4 enzymes was made at 30°C, and aeration was performed at 200 rpm.

After culturing as described above in the culture medium containing the substrate, the supernatant in which the bacteria and the synthesized melanin-digo were dissolved in the culture medium were separated by centrifugation. The pH of the separated supernatant was lowered to 2.0 by adding hydrochloric acid to precipitate melanin-digo. When all the synthesized melanin-digo was precipitated, it was recovered by centrifugation.

comparative example One: melC L- using overexpressed E. coli from tyrosine synthesis of melanin

[Scheme 2]

melC overexpressing strain, Escherichia while supplying L-tyrosine coli BL21(DE3):: melanin was biosynthesized by culturing melC. A specific synthesis mechanism is shown in Scheme 2 above. After L-tyrosine was converted into a dihydroxyindole (DHI)-derived material by melC, it was polymerized by bonding on a benzene ring through a catechol structure included therein.

comparative example 2: CYP102G4 Synthesis of indigo from L-tryptophan using overexpressed E. coli

[Scheme 3]

CYP102G4 overexpressing strain, Escherichia while supplying L-tryptophan coli BL21(DE3):: cyp102G4 was cultured to biosynthesize indigo. A specific synthesis mechanism is shown in Scheme 3 above. The CYP102G4 mediated the hydroxylation reaction at the C-2 position of the indole ring obtained from L-tryptophan to form 2-hydroxyindole. Then, as in the above scheme, the pyrrole ring was dimerized to form a final indigo molecule. The indigo molecules existed as individual molecules upon completion of the reaction without further polymerization.

Experimental example 1: synthesized melanindigo's Sympathy

The chemical composition of the melanin-digo synthesized according to Example 1 was analyzed by 15T FT-ICR MS (15 tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometer), and the result was analyzed according to Comparative Example 1 of the conventional melanin, tyrosine melanin. It is shown in FIG. 1 by comparison with the chemical composition. As shown in FIG. 1 , it was confirmed that the melanindigo of Example 1 had a chemical composition completely different from that of tyrosine-derived melanin (eumelanin). Specifically, in the conventional tyrosine melanin, the ratio of CHO was significantly higher than that of CHONS, but in the case of melanindigo, the ratio of CHO decreased, while the ratio of CHOS and CHONS was relatively increased.

Furthermore, the chemical bond between melanindigo and tyrosine melanin was analyzed and shown in Table 1 below. As shown in Table 1, in the case of melanindigo, the degree of unsaturation, ie, the ratio of double bonds, increased in DBE (double bond equivalent) analysis, and the aromaticity index (AI), which is an index of aromaticity, also increased. On the other hand, the ratio of oxygen atoms and hydrogen atoms to carbon atoms, respectively, O/C and H/C, respectively, decreased significantly, while the ratio of nitrogen atoms to carbon atoms, N/C, increased significantly by more than six times. These results show that the chemical composition of melanindigo, a novel polymer according to the present invention, has increased condensation due to increased unsaturation and decreased O/C ratio, and the significantly increased N/C ratio includes nitrogen-based compounds in the structure. will indicate

assigned peak DBE AI O/C H/C N/C S/C Tyrosine Melanin 388 9.25 0.21 0.32 1.24 0.0208 0.0136 melanindigo 1716 9.63 0.32 0.25 0.78 0.1234 0.01254

In addition, by synthesizing the obtained data, the molecular distribution diagram of melanin-digo and tyrosine-melanin was plotted, and the results are shown in FIG. 2 . As shown in FIG. 2 , the type of tyrosine melanin was shown to have a chemical composition and structure of an unsaturated hydrocarbon series or a lipid series, whereas melanindigo is a complex containing lignin type aromatics. (complex) It was confirmed that the structure had a more complex structure from the distribution (red dots).

Experimental example 2: Color analysis

The color of the synthesized melanin-digo was visually confirmed, and it is shown in FIG. 3 together with indigo and melanin, which are comparative examples. As shown in FIG. 3 , melanindigo synthesized according to Example 1 exhibited a significantly darker color than indigo and melanin biosynthesized by a similar method.

For a more specific analysis, the fine particle shape was confirmed through the SEM image of the synthesized melanin (Comparative Example 1) and the melanin-digo of the present invention (Example 1), and the degree of coloring was confirmed by staining with cellulose. It was. As shown in FIG. 4, it was confirmed that the melanindigo according to the present invention was arranged in a clearer polymer structure than that of melanin, and the color development was also remarkably improved when dyeing for cellulose.

Furthermore, the RGB color of the melanin-digo of the present invention was extracted and compared with the results for melanin, as shown in FIG. 5 . As shown in Figure 5, the conventional melanin showed a brown-based color with a relatively high ratio of red and green, whereas melanindigo showed a color close to black with a reduced ratio of red and green.

Claims (10)

Compared to eumelanin derived from tyrosine, it exhibits increased double bond equivalent (DBE), and aromaticity index (AI); The CHON and CHO ratios decreased to levels of 90 to 93%, and 50%, respectively, and the CHOS and CHONS ratios increased to levels of 600%, and 800% or more, respectively, indicating reduced O/C and H/C ratios, A complex exhibiting an increased N/C ratio of more than 6 fold.
According to claim 1,
The 15T FT-ICR MS (15 tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometer) graph shows 1716 assigned peaks, with DBE of 9.63, AI of 0.32, O/C of 0.25, H/C of 0.78, and H/C of 0.1234. N/C, and an S/C of 0.01254.
culturing a strain overexpressing CYP102G4 and melC while providing tyrosine,
Compared to eumelanin derived from tyrosine, it exhibits increased double bond equivalent (DBE), and aromaticity index (AI); The CHON and CHO ratios decreased to levels of 90 to 93%, and 50%, respectively, and the CHOS and CHONS ratios increased to levels of 600%, and 800% or more, respectively, indicating reduced O/C and H/C ratios, Method for producing a complex exhibiting an N/C ratio increased by more than 6 times.
4. The method of claim 3,
The strain overexpressing CYP102G4 and melC is Escherichia Recombinant strain Escherichia prepared by introducing foreign genes for melC and CYP102G4 expression into coli BL21(DE3) coli BL21(DE3):: melC :: cyp102G4 , the production method.
4. The method of claim 3,
The culture is to be achieved using a culture medium based on LB medium (luria-bertani broth), the manufacturing method.
6. The method of claim 5,
The culture medium is a heme precursor necessary for CYP102G4 protein expression, CuSO ₄ and 5-aminolevulinic acid (ALA), which are coenzymes necessary for melC expression, are additionally included. .
6. The method of claim 5,
The culture medium will further comprise tryptone, yeast extract (yeast extract) and sodium chloride, the manufacturing method.
A dye composition comprising the compound of claim 1 .
9. The method of claim 8,
A composition that is non-toxic.
9. The method of claim 8,
A composition for dyeing paper, fibers or fabrics.

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