KR20200140636A - Novel indigoid polymer and biosynthesis thereof - Google Patents

Abstract

The present invention relates to a novel indigo-based polymer not toxic to the human body, to a biosynthetic method thereof, and to a use and, more specifically, to a compound represented by chemical formula 1.

Description

Novel indigoid 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 by themselves, since the binding with fibers is weak and color development is difficult, they are sometimes dyed with a mordant such as alum, which is concerned with water.

Prior to the development of chemistry as today, ferric oxide (Fe ₂ O ₃ ) was used for coloring, or pigments from insects and plants such as roosters, which are a kind of shellfish, were extracted and used as dyes. Natural dyes are divided into animal dyes, vegetable dyes, and mineral dyes, depending on the method of collecting the dyes obtained from nature. It wasn't until the middle of the 19th century that it was possible to produce various colors by synthesizing colored organic substances (dyes) without relying on natural pigments. In this era, the so-called modern industry sprang up, and the acid/alkali industry and the iron-making industry using coke took place, and in the UK, coal was dried to supply city gas.

Under this background, while coke for steelmaking was dried and research on coal tar, the remaining residue, in the process of synthesizing kinine from toluidine, a component of coal tar, we accidentally discovered a vividly colored pigment, which makes silk beautiful. It was named mauvein because it can be colored purple. Later, the dye was improved and a synthetic dye company called Mobra was established. Furthermore, as research on the structure and synthesis method of aromatic compounds centered on benzene progressed, basic theories about color development such as chromophores and chromophores appeared. The synthesis of dyes has gradually developed thanks to such basic chemical research, development of synthetic 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 madders, and has been used to dye cotton and other red from ancient times. Later, it was discovered that alizarin was an anthracene derivative, and in 1868 it was successfully synthesized from anthracene. From this, mass production of Alizarin, which does not depend on natural ingredients, has become possible.

Indigo, a pigment extracted from indigo, has been used in Egypt since about 2,000 to 1,000 BC, being well stained with animal island fibers such as wool or silk or vegetable fibers such as cotton or hemp. Indigo is dyed with a beautiful indigo color and has good weather resistance. Indigo, a raw material, was cultivated in large quantities mainly in India, and was synthesized by German buyers in 1880. Subsequently, the use of natural indigo decreased significantly, and the cultivation of indigo declined rapidly as the synthetic method was improved to allow simple synthesis.

The synthesis of these two dyes was a dramatic shift from a conventional nature-dependent method to a chemically synthesized method. Since then, the length of chemical synthesis has rapidly expanded, and furthermore, as a result of research on synthetic dyes centered on the tar industry in the mid-19th century, not only tens of thousands of types of synthetic dyes have been developed, but also the foundation for the synthetic pharmaceutical industry has been established. Was done.

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

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

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 carbon involved therein as 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 the O

Is a double bond,

It is polymerized with neighboring monomers through at least one site 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 heteropolymerization of one or more monomers selected from compounds represented by the following 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 with 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 by homogeneous and heterodimerization thereof, three kinds of monomers represented by Chemical 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 may be provided. 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 that overexpresses 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 carbon involved therein as 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 the O

Is a double bond,

It is polymerized with neighboring monomers through at least one 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 that overexpresses CYP102G4 and melC used in the production method of the present invention is Escherichia recombinant strain prepared by introducing a foreign gene for the expression melC and CYP102G4 coli BL21 (DE3), Escherichia coli BL21(DE3):: may be melC :: cyp102G4 . The strain can be prepared by using, without limitation, genetic information and/or genetic recombination methods known in the art.

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

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

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

For example, the cultivation of the strain may be performed at 37°C, and the enzyme, that is, overexpression of CYP102G4 and melC may be achieved at 30°C, but is not limited thereto, and conditions and/or enzymes suitable for normal strain cultivation In consideration of the conditions required for overexpression of, those skilled in the art can appropriately adjust. In addition, during the culture, it may be ventilated 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 the formula (1).

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, fibers or fabrics, but is not limited thereto. Furthermore, these dyed papers, fibers and fabrics are items mainly used for packaging materials, clothes or jewelry, etc., and not toxic to the human body is one of the important factors that the dye composition used therein must have.

The dye composition of the present invention may further include additives such as a solubilizing agent, a buffering agent, and an antifreezing agent. The solubilizing agent is an additive component that prevents agglomeration of the dye and helps dissolution, and representative examples include urea, thiourea, caprolactam, and the like. The buffering agent is a component that serves to buffer pH in order to prevent decomposition of dyes during storage and storage, and representative examples include sodium phosphate, potassium phosphate, sodium acetate, and the like. The anti-icing agent is a component that serves to prevent the liquid dye from freezing by lowering the freezing point during liquid freezing storage, and representative examples thereof include ethylene glycol, polyethylene glycol, and polyhydric alcohol compounds. At this time, the additive may be added in the range of about 1 to 20% by weight based on the total weight of the composition, but is not limited thereto.

The novel indigo-based polymer of the present invention is a material synthesized from tyrosine, an amino acid by biosynthesis by enzyme action, so it is not toxic to the human body, and has superior color development and/or coloring power compared to conventional indigo or melanin, making it 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 the melanindigo of the present invention by 15T FT-ICR mass spectrometry.
2 is a diagram showing a molecular distribution diagram of tyrosine melanin according to Comparative Example 1 and melanindigo of the present invention.
3 is a diagram showing the difference in color 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.
Figure 4 is a diagram showing the (left) SEM image of melanin synthesized by melC overexpressing E. coli, and melanindigo synthesized by melC and CYP102G4 overexpressing E. coli, and (right) 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 more specifically, and the scope of the present invention is not limited by these examples.

Example One: melC And CYP102G4 L- using overexpressing E. coli From tyrosine Synthesis of indigoide-based melanin

[Scheme 1]

While supplying the L- tyrosine melC and CYP102G4 overexpression strain, for example, by introducing a foreign gene for the expression CYP102G4 melC and Escherichia coli BL21 (DE3) is prepared by a recombinant strain, Escherichia Indigo was biosynthesized by culturing coli BL21(DE3):: melC :: cyp102G4 . The specific synthesis mechanism is shown in Scheme 1. Specifically, by dimerization using a hydroxy group introduced on the pyrrole ring of an indole derivative obtained as an intermediate by CYP102G4 to form a compound containing an indigoid structure, a catechol structure formed by melC Through polymerization, an indigo polymer, which is 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 Heme precursor, 5-aminolevulinic acid (ALA), which is a precursor necessary for protein expression, CuSO ₄ as a coenzyme required for melC expression, and L-tyrosine as a substrate were added thereto. Specifically, a 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 above culture medium, but overexpression of the melC and CYP102G4 enzymes was performed at 30° C., and aeration was performed at 200 rpm.

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

Comparative example One: melC L- using overexpressing E. coli From tyrosine Synthesis of melanin

[Scheme 2]

Escherichia , a strain that overexpresses melC while supplying L-tyrosine coli BL21(DE3):: melC was cultured to biosynthesize melanin. The specific synthesis mechanism is shown in Scheme 2. L-tyrosine was converted to a material derived from dihydroxyindole (DHI) by melC, and then polymerized by bonding on a benzene ring through a catechol structure contained therein.

Comparative example 2: CYP102G4 Synthesis of indigo from L-tryptophan using overexpressing E. coli

[Scheme 3]

Escherichia , a strain that overexpresses CYP102G4 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 formed 2-hydroxyindole by mediating a hydroxylation reaction at the C-2 position of the indole ring obtained from L-tryptophan. Thereafter, as in the reaction 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 Sympathy

The chemical composition of the melanindigo synthesized according to Example 1 was analyzed by 15T FT-ICR MS (15 tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometer), and the results were analyzed for tyrosine melanin, a conventional melanin prepared according to Comparative Example 1. It is shown in Figure 1 in 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. Specifically, in the conventional tyrosine melanin, the ratio of CHO was significantly higher than that of CHONS, whereas the ratio of CHO decreased in the case of melanindigo, whereas the ratio of CHOS and CHONS was relatively increased.

Further, 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, that is, the ratio of double bonds, increased in the double bond equivalent (DBE) analysis, and the aromaticity index (AI), which is an index of aromatics, also increased. On the other hand, the ratio of oxygen and hydrogen atoms to carbon atoms, O/C and H/C, respectively, decreased significantly, while the ratio of nitrogen atoms to carbon atoms and N/C increased significantly more than 6 times. These results indicate that the chemical composition of melanindigo, a novel polymer according to the present invention, increased condensation due to increased unsaturation and decreased O/C ratio, and the significantly increased N/C ratio contained nitrogen-based compounds in the structure. Is to represent.

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, the obtained data were synthesized to plot the molecular distribution of melanindigo and tyrosine melanin, and the results are shown in FIG. 2. As shown in Figure 2, the type of tyrosine melanin is shown to have a chemical composition and structure of an unsaturated hydrocarbon series or a lipid series, whereas melanindigo is a complex containing an aromatic in the form of lignin. It was confirmed that the (complex) structure had a more complex structure from the distribution (red dot).

Experimental example 2: color analysis

The color of the synthesized melanindigo was visually confirmed, and it is shown in FIG. 3 along with comparative examples of indigo and melanin. As shown in FIG. 3, melanindigo synthesized according to Example 1 exhibited a significantly darker color than indigo and melanin biosynthesized in a similar manner.

For a more specific analysis, the microparticle shape was confirmed through SEM images of the synthesized melanin (Comparative Example 1) and the melanindigo of the present invention (Example 1), and the degree of coloration was confirmed by dyeing cellulose, as shown in FIG. Done. As shown in FIG. 4, it was confirmed that melanindigo according to the present invention was arranged in a more clear polymer structure than melanin, and color development was also significantly improved when dyeing cellulose.

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

Claims (10)

Compound represented by the following formula (1):
[Formula 1]

In Formula 1,

Is determined to satisfy the valence of carbon involved therein as 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,
Connected to the O

Is a double bond,
It is polymerized with neighboring monomers through at least one site of L ₁ to L ₄ ,
n is an integer from 2 to 10,000.
The method of claim 1,
The polymer represented by Formula 1 is a polymer formed by homogeneous or heteropolymerization of at least one monomer selected from compounds represented by the following Formulas 2 to 4:
[Formula 2]

[Formula 3]

[Formula 4]

.
Comprising the step of culturing a strain that overexpresses CYP102G4 and melC while providing tyrosine,
A method of preparing a compound represented by the following Formula 1:
[Formula 1]

In Formula 1,

Is determined to satisfy the valence of carbon involved therein as 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,
Connected to the O

Is a double bond,
It is polymerized with neighboring monomers through at least one 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.
The method of claim 3,
The strain overexpressing CYP102G4 and melC is Escherichia recombinant strain prepared by introducing a foreign gene for the expression melC and CYP102G4 coli BL21 (DE3), Escherichia coli BL21(DE3):: melC :: cyp102G4 that is, the manufacturing method.
The method of claim 3,
The cultivation is to be achieved using a culture medium based on LB medium (luria-bertani broth).
The method of claim 5,
The culture medium is a heme precursor required for CYP102G4 protein expression, a coenzyme required for melC expression, CuSO ₄ and 5-aminolevulinic acid (ALA), which additionally contains .
The method of claim 5,
The culture solution further comprises tryptone, yeast extract, and sodium chloride.
A dye composition comprising the compound of claim 1.
The method of claim 8,
A composition that is non-toxic.
The method of claim 8,
A composition for dyeing paper, fibers or fabrics.

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