JP2006158304A - Method for producing melanin precursor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently producing a melanin precursor. <P>SOLUTION: This method for producing the melanin precursor comprises a process for converting at least one substrate compound selected from the group consisting of tyrosine and its analogues in the presence of cells exhibiting high catechol oxidase activity due to one or more factors of the following (a) to (d) and a process for recovering the melanin precursor. (a) A gene encoding a polypeptide having catechol oxidase activity is expressed in the presence of a higher activity promoter than that of an originally expressed promoter. (b) The cells each has a plurality of gene copies encoding the polypeptide having the catechol oxidase activity. (c) The cells each exhibits high catechol oxidase activity, because of having a gene encoding a polypeptide having a mutated catechol oxidase activity. (d) The cells are subjected to a tyrosinase activation treatment to exhibit a high catechol oxidase activity. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for producing a melanin precursor added to cosmetics, glasses, plastics, foods and the like.

Melanin is a yellow to black pigment widely present in animals and plants, and is known to have an ultraviolet absorption function, a radical capture function, an antioxidant function, and the like. Melanin is widely used as an additive for cosmetics, foods and the like because it is a biologically derived substance and has high safety.

  For example, melanin is used to give an ultraviolet absorbing function to sunscreen creams, sunglasses, and the like. It is also used as an antioxidant for foods and plastics. Furthermore, it is also added to white hair dyeing as a pigment.

  Thus, since melanin is a very useful substance, the manufacturing method has been variously examined. Conventional melanin production methods are roughly classified into extraction of melanin existing in nature, chemical synthesis, and enzymatic reaction using tyrosinase.

For example, melanin can be extracted and purified from cultures of Aureobasidium microorganisms, Aspergillus microorganisms, and Streptomyces microorganisms that produce melanin in a relatively large amount. However, the amount of melanin produced by wild-type microorganisms is limited, and it is difficult to produce a practically sufficient amount. Moreover, since tyrosinase belonging to the genus Streptomyces is secreted, the cultured cells cannot be reused and the efficiency is poor.
A method of extracting from a squid ink bag is also known, but melanin in a squid ink bag is said to have poor solubility.
Although it is possible to chemically synthesize tyrosine or the like as a raw material, it requires a strong oxidation reaction and requires a complicated and expensive technology. A method of oxidizing tyrosine in hydrogen peroxide and synthesizing melanin through a polymerization reaction is also known, but the obtained melanin is insoluble in water and is difficult to use as cosmetics or dyes.
As shown in FIG. 1, in vivo, melanin pigments are dopachromes produced by catalyzing the oxidation of tyrosine or 3,4-dihydroxyphenylalanine (DOPA), which is a melanin-producing enzyme, tyrosinase, Biosynthesized by polymerization of monomers such as 6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid. The melanin pigment produced in this way is present in small particles in melanin-producing cells such as skin and hair, is a polymer compound that is insoluble in water, soluble in hot concentrated sulfuric acid and strong alkali, Its structure is not clear.

Attempts have also been made to produce melanin by polymerizing monomers produced via dopaquinone by allowing tyrosinase to act on the substrate tyrosine or DOPA. As tyrosinase, tyrosinase preparations purified from mushrooms and tyrosinase preparations isolated from mouse and hamster cells are commercially available.
However, the melanin pigment produced using tyrosinase is insoluble in water and quickly precipitates. When used as dyes for fibers, leather, etc., insoluble polymer pigments cannot penetrate tissues and cannot dye objects.
Therefore, in order to use melanin as a dye or the like, it is necessary to devise a technique for penetrating it into the object to be dyed.

  An object of the present invention is to provide an efficient method for producing a melanin precursor.

In order to solve the above-mentioned problems, the present inventor repeated research and obtained the following knowledge.
(1) Since melanin is a polymer that is hardly soluble in water, for example, when used as a dye, it is difficult to permeate into an object, but in the form of a mixture of melanin constituent monomers (melanin precursor) that is a polymer. If used as a dye, it can easily penetrate into the target article. Furthermore, it can be easily polymerized in the object to be dyed to produce melanin.
(2) Catechol oxidase has high affinity for at least one substrate compound selected from the group consisting of L-tyrosine, D-tyrosine, L-DOPA, D-DOPA and their analogs, but it is an oxidation product thereof. Low affinity for some dopachromes. That is, before the non-enzymatic oxidation of dopachrome, which is an enzyme reaction product, proceeds by allowing cells exhibiting an excessive amount of catechol oxidase activity or an enzyme exhibiting an excessive amount of catechol oxidase activity to act on the substrate compound. By rapidly enzymatically oxidizing the substrate compound, the melanin precursor can be efficiently accumulated.
(4) Adjust the pH of the dopachrome solution to 5 to 7 and store this solution in the presence of oxygen while maintaining the pH, or adjust the pH to 3 to 5 in the absence of oxygen. By storing the solution while maintaining the pH or adjusting the pH to 5 to 10 and storing the solution while maintaining the pH in the absence of oxygen, 5,6-dihydroxyindole can be efficiently produced. it can. That is, dopachrome is converted to 5,6-dihydroxyindole by spontaneous decarboxylation, but the present inventor has found an efficient method for converting dopachrome to 5,6-dihydroxyindole.
(5) By adding a water-soluble organic solvent to the dopachrome solution and storing it, dopachrome is efficiently converted into 5,6-dihydroxyindole. However, when a basic water-soluble organic solvent such as aminoethanol is used, it is efficiently converted to 5,6-dihydroxyindolecarboxylic acid and can be stored stably.
(6) By adding salt to the dopachrome solution and storing it, dopachrome is efficiently converted to 5,6-dihydroxyindole.
(7) By adding divalent copper ions to the dopachrome solution and storing it, dopachrome is converted into 5,6-dihydroxyindole-2-carboxylic acid efficiently and non-enzymatically.
(8) A buffer, especially a phosphate buffer, is added to the dopachrome solution and stored at a low temperature of about −80 to −0 ° C., so that non-enzymatically efficient dopachrome is 5,6-dihydroxyindole-2. -Converted to carboxylic acid.

  The present invention has been completed based on the above findings, and provides the following method for producing a melanin precursor.

1. In the reaction solution, at least one substrate compound selected from the group consisting of tyrosine, DOPA and analogs thereof, and one or more factors of (a), (b), (c) and (d) below: An oxidation step of oxidizing a substrate compound and converting it into a melanin precursor in the presence of cells exhibiting high catechol oxidase activity in
The manufacturing method of a melanin precursor including the collection | recovery process which collect | recovers a melanin precursor from a reaction liquid.
(a) This gene is expressed under a promoter having a higher activity than a promoter that originally expresses a gene encoding a polypeptide having catechol oxidase activity.
(b) It has multiple copies of a gene encoding a polypeptide having catechol oxidase activity.
(c) High catechol oxidase activity is exhibited by having a mutated gene encoding a polypeptide having catechol oxidase activity.
(d) High catechol oxidase activity is exhibited by tyrosinase activation treatment.

2. Item 2. The method according to Item 1, wherein the catechol oxidase activity of the cell when L-DOPA is used as a substrate is 0.1 U / OD ₆₀₀ or more.
3. Item 3. The method according to Item 1 or 2, wherein the catechol oxidase activity when 1 mol of L-DOPA is used as a substrate is 5 × 10 ⁵ U / mol or more.
4). Item 4. The method according to Item 1, 2, or 3, wherein the polypeptide having catechol oxidase activity is tyrosinase.

  5. Filamentous tyrosinase selected from the group consisting of Aspergillus fungi, Neurospora fungi, Rhizomucor fungi, Tricoderma fungi, and Penicillium fungi Item 5. The method according to Item 4, which is a fungal tyrosinase.

  6). Item 6. The method according to Item 5, wherein the tyrosinase is a polypeptide encoded by the melB tyrosinase gene, melD tyrosinase gene or melO tyrosinase gene of Aspergillus oryzae.

  7). Item 7. The method according to any one of Items 1 to 6, wherein the cell is a microbial cell selected from the group consisting of Escherichia coli, yeast and filamentous fungi.

  8). Item 1 to 7 further comprising a composition adjustment step (A) for adjusting the composition by storing the melanin precursor obtained by the oxidation step in the presence of divalent copper ions between the oxidation step and the recovery step. The method in any one of.

  9. Furthermore, a composition adjustment step (B) for adjusting the composition by storing the melanin precursor obtained by the oxidation step in the presence of a buffer at −80 to 0 ° C. is included between the oxidation step and the recovery step. Item 9. The method according to any one of Items 1 to 8.

  Ten. Furthermore, the composition adjustment process (C) which adjusts the composition by storing the melanin precursor obtained by the oxidation process in the presence of a salt between the oxidation process and the recovery process, The method described in 1.

  11． Furthermore, between the oxidation step and the recovery step, the melanin precursor obtained in the oxidation step is maintained at pH 3-5 in the absence of oxygen, pH 5-10 in the absence of oxygen, or pH 5-7 in the presence of oxygen. Item 11. The method according to any one of Items 1 to 7 and 10, which comprises a composition adjustment step (D) for adjusting the composition by storage.

  12． Furthermore, between the oxidation step and the recovery step, the melanin precursor obtained by the oxidation step includes a composition adjustment step (E) for adjusting the composition by storing the melanin precursor in the presence of a water-soluble organic solvent. The method according to any one of 7, 10 and 11.

13. When at least one substrate compound selected from the group consisting of tyrosine, DOPA, and analogs thereof is used, an enzyme having catechol oxidase activity has a catechol oxidase activity of 5 × when 1 mol of L-DOPA is used as a substrate. An oxidation step of oxidizing the substrate compound to convert it into a melanin precursor by acting so that it becomes 10 ⁵ U / mol or more;
The manufacturing method of a melanin precursor including the collection | recovery process which collect | recovers a melanin precursor from a reaction liquid.

  14. Item 14. The method according to Item 13, wherein the polypeptide having catechol oxidase activity is tyrosinase.

  15． Item 13 or 14 further includes a composition adjustment step (A) in which the melanin precursor obtained by the oxidation step is stored in the presence of divalent copper ions to adjust the composition between the oxidation step and the recovery step. The method described in 1.

  16． Furthermore, a composition adjustment step (B) for adjusting the composition by storing the melanin precursor obtained by the oxidation step in the presence of a buffer at −80 to 0 ° C. is included between the oxidation step and the recovery step. Item 16. The method according to Item 13, 14 or 15.

  17． Item 13. The method according to Item 13 or 14, further comprising a composition adjustment step (C) for adjusting the composition by storing the melanin precursor obtained by the oxidation step in the presence of a salt between the oxidation step and the recovery step. Method.

  18． Furthermore, between the oxidation step and the recovery step, the melanin precursor obtained in the oxidation step is maintained at pH 3-5 in the absence of oxygen, pH 5-10 in the absence of oxygen, or pH 5-7 in the presence of oxygen. Item 18. The method according to Item 13, 14 or 17, comprising a composition adjustment step (D) for adjusting the composition by storage.

  19. Furthermore, the composition adjustment step (E) for adjusting the composition by storing the melanin precursor obtained by the oxidation step in the presence of a water-soluble organic solvent between the oxidation step and the recovery step, The method according to 14, 17 or 18.

  20． Item 20. A melanin precursor obtained by the method according to any one of items 1 to 19.

  According to the present invention, an efficient method for producing a melanin precursor is provided. Furthermore, according to the method of the present invention, it is possible to efficiently produce a mixture of melanin constituent monomers (melanin precursor) by suppressing the production of melanin due to polymerization of the melanin precursor.

  Further, according to the method of the present invention, the melanin constituent monomer can be produced easily and inexpensively, and therefore the method of the present invention is suitable for industrial production of melanin precursors.

Since melanin is a poorly water-soluble polymer, it is difficult to stain organic matter, but since melanin precursor is water-soluble, it penetrates the melanin precursor into the object to be dyed, and then polymerizes the object efficiently. can do.
Among the melanin precursors, indoles have a bactericidal action, so that a bactericidal effect can be expected simultaneously with the dyeing. Furthermore, since the reactivity is high, not only organic substances but also metals such as stainless steel can be dyed.
Moreover, the melanin precursor of the composition used as the melanin of a specific polymerization state (for example, planarly polymerized melanin) can also be manufactured by setting reaction conditions.

(I) Method for producing first melanin precursor
Basic Structure The first method for producing a melanin precursor of the present invention exhibits at least one substrate compound selected from the group consisting of tyrosine, DOPA and analogs thereof and high catechol oxidase activity in the reaction solution. An oxidation step of oxidizing a substrate compound to convert it into a melanin precursor in the presence of cells; and a step of recovering the melanin precursor from the reaction solution.
Oxidation process of substrate compound
<Polypeptide having catechol oxidase activity>
Catechol oxidase activity is activity that catalyzes the production of o-quinone by oxidation of catechol. Polypeptides having catechol oxidase activity (hereinafter abbreviated as “catechol oxidase”) include enzymes called catechol oxidase, monophenol oxidase, diphenol oxidase, o-diphenolase, tyrosinase, and the like. These usually have monophenol oxidase activity. In addition, although it does not have monophenol oxidase, laccase and peroxidase having polyphenol oxidase activity are also included in the polypeptide having catechol oxidase activity. Among catechol oxidases, it is preferable to use tyrosinase from the viewpoint that a natural melanin precursor can be efficiently produced because of its high affinity for L-DOPA.

Catechol oxidase may be an enzyme derived from any organism, but tyrosinase derived from filamentous fungi is particularly preferable because of its high expression efficiency and stability in the host cell. Examples of such filamentous fungi include Aspergillus sp., Neurospora sp., Rhizomucor sp., Trichoderma sp., Penicillium sp. Among them, tyrosinase of Aspergillus filamentous fungi is preferable because it is relatively stable against heat and has been confirmed to be safe. Specifically, Aspergillus oryzae melB gene (Japanese Patent Laid-Open No. 2002-191366) Gazette), melD gene (Japanese Patent Application No. 2002-373069) or melO gene (Molecular cloning and nucleotide sequence of the protyrosinase gene, melO, from Aspergillus oryzae and expression of the gene in yeast cells. Biochim Biophys Acta. 1995 Mar 14; 1261 ( 1): Tyrosinase encoded by 151-154.) Can be mentioned.
<Cell>
As the cells showing high catechol oxidase activity, the following cells (a), (b), (c) and / or (d) are used.
(a) This gene is expressed under a promoter having a higher activity than a promoter that originally expresses a gene encoding a polypeptide having catechol oxidase activity.
(b) It has multiple copies of a gene encoding a polypeptide having catechol oxidase activity.
(c) High catechol oxidase activity is exhibited by having a mutated gene encoding a polypeptide having catechol oxidase activity.
(d) High catechol oxidase activity is exhibited by tyrosinase activation treatment.

  Specifically, for example, a catechol oxidase gene cloned into a vector usually used for mass expression of a protein is introduced into a host cell, and the host cell is incorporated into a host chromosome or cultured in a host cell having this in a plasmid state. By doing so, a large amount of catechol oxidase can be produced. Thus, this gene may be expressed under a promoter that is more active than the promoter that originally expresses the gene.

  In addition, for example, a diploid or higher cell in which a plurality of catechol oxidase genes may be retained may be used. In addition, for example, since there are triploid and tetraploid cells in practical yeasts, these can be preferably used. In this way, a cell exhibiting high catechol oxidase activity can be obtained by increasing the copy number from that of the gene.

  Furthermore, cells having increased catechol oxidase activity due to mutation of the catechol oxidase gene or cells into which such a mutated catechol oxidase gene has been introduced can be used. Thus, it can be set as the cell which shows high catechol oxidase activity by setting it as the variant enzyme which shows higher activity than a natural type enzyme.

  As will be described later, a cell having enhanced catechol oxidase activity by performing a treatment for coordinating divalent copper ions or a treatment with an acidic solution of about pH 2.8 to 3.2 can also be used.

  Among them, a cell in which this gene is expressed under a promoter having a higher activity than a promoter that originally expresses a gene encoding a polypeptide having catechol oxidase activity (a) is preferable.

Although the kind of cell which produces catechol oxidase is not specifically limited, It is preferable that it is a microorganism at the point which mass culture is easy. Examples of microorganisms that are easy to use include Escherichia coli, yeast, and filamentous fungi. Among them, it is preferable to use yeast because it is safe, has high production efficiency of catechol oxidase, is a single cell, and has a high cell sedimentation rate, so that the cells after reaction can be separated by centrifugation at a relatively low speed. . Among these, Saccharomyces cerevisiae is preferable in that the bacterial cell is robust, and thus the outflow of the protein derived from the bacterial cell into the reaction solution is suppressed and the genetic manipulation is easy.
In order for catechol oxidase, in particular tyrosinase, to exhibit activity, it is necessary to coordinate a divalent copper ion to the catalytic activity center. Since the expression level of catechol oxidase is small in wild type cells, it is sufficiently active due to the coordination of divalent copper ions present in the cells, but is sufficient for cells in which the expression level of catechol oxidase is improved by transformation or the like. Catechol oxidase activity may not be obtained. Accordingly, when any of the above cells (a) to (d) is used, it is preferable to coordinate the divalent copper ion to the catalytically active center of catechol oxidase by treating the cell with divalent copper ion in advance. . Specifically, the transformant is suspended in, for example, a copper sulfate solution of about 0.1 to 2 mM and allowed to stand at about 30 to 40 ° C. for about 0.5 to 2 hours to sufficiently divalent copper for intracellular catechol oxidase. Ions can be coordinated.
In addition, when using any of the above cells (a) to (d), among catechol oxidases, tyrosinase, in particular tyrosinase derived from Aspergillus oryzae, is matured by treatment with an acidic solution of about pH 2.8 to 3.2. And activate. Therefore, for example, by suspending in a sodium acetate buffer solution (pH = 3) of about 20 to 200 mM and allowing to stand at about 0 to 40 ° C. for about 0.5 to 1 hour, the catechol oxidase activity can be further improved.
When cells are yeast, when L-DOPA is used as a substrate, cells having catechol oxidase activity of 0.1 U / OD ₆₀₀ or more, preferably 0.5 U / OD ₆₀₀ or more, more preferably 1 U / OD ₆₀₀ or more are used. It is preferable. In the case of cells other than yeast, cells having similar catechol oxidase activity may be used. The upper limit of the catechol oxidase activity of the cell is not particularly limited, but is usually about 5 U / OD ₆₀₀ .

Furthermore, when 1 mol of L-DOPA is used as a substrate, the concentration is usually 5 × 10 ⁵ U / mol or more, preferably 5 × 10 ⁶ U / mol or more. The upper limit of the catechol oxidase activity per 1 mol of L-DOPA is not particularly limited, but usually about 5 × 10 ⁷ U / mol is sufficient, and more than that is expensive.
In the present invention, the catechol oxidase activity of cells and the catechol oxidase activity of enzymes are activities measured by the methods described in the Examples.

  In the method of the present invention, by using a cell exhibiting high catechol oxidase, the catechol oxidase is excessively present in the cell with respect to the substrate. Thereby, the melanin precursor produced | generated from the substrate compound further polymerizes, and a melanin precursor produces | generates from a substrate compound faster than the speed | rate which melanin produces | generates, and a melanin precursor accumulate | stores efficiently in a reaction system.

For the cells, for example, those immobilized by a known method such as a carrier binding method, a inclusion method, a crosslinking method, or a photocrosslinking method can be used. It becomes possible to separate from the reaction system.
<Promoter>
When a large amount of catechol oxidase is produced by using a highly active promoter, the promoter is not particularly limited as long as it is a promoter having a higher activity than the promoter that originally expresses this gene. A promoter suitable for the host to be used may be used.

  For example, when E. coli is used as a host, examples include T7 promoter, tac promoter, tacI promoter, lacZ promoter, trp promoter, BAD promoter, lac promoter, λPL promoter, spa promoter, sp6 promoter, lpp promoter, and the like. Of these, the T7 promoter, tac promoter, and trp promoter are preferable, and the T7 promoter is more preferable.

  When yeast is used as the host, SED1 promoter, ADH1 promoter, PGK promoter, GAPDH promoter, TDH1 promoter, PHO5 promoter, GAL4 promoter, GAL10 promoter, CUP1 promoter and the like can be mentioned. Among these, SED1 promoter, ADH1 promoter, PGK promoter, and GAPDH promoter are preferable, and SED1 promoter is more preferable.

When gonococcus is used as a host, melO promoter, glaA promoter, manganese superoxide dismutase promoter (sodM promoter), copper, zinc superoxide dismutase promoter, cytochrome P450 promoter, catalase A promoter, catalase B promoter, small V-ATPase Promoter, actin promoter, histone H2 promoter (described above, disclosed in JP-A-2001-224381); cruciform binding protein promoter, peptidylprolyl cis-trans isomerase promoter, ubiquitin conjugating enzyme promoter, ATP-dependent transporter promoter, H + trans Porting ATP synthase promoter, ubiquitin promoter, Rab5-like GTPase promoter, ribosomal protein L37 promoter, ribosomal protein S16 promoter, phosphatidyl synthetase promoter, mRNA removal factor I 25 kDa subunit promoter, histone H3 subunit promoter, 26S proteasome p44.5 protein promoter, yeast-derived starvation survival protein promoter, putative membrane Protein promoter, lipid transporter POX18-like protein promoter, peroxisome membrane protein per10-like protein promoter, vacuolar H + ATPase subunit promoter, DNA binding P52 / P100 complex 100 kDa subunit promoter, adenosine kinase promoter, replication factor A-like protein promoter , T. reesei-like protein kinase promoter, calcium-binding protein promoter, mannose-binding protein promoter, sorbit Rule utilization protein promoter, thioredoxin promoter, cytochrome C promoter, mannose lectin promoter, manganese superoxide dismutase 2 promoter, sar1 promoter, d12-desaturase promoter, d9-desaturase promoter, d6-desaturase promoter, alcohol acyltransferase promoter, ccg- 9 promoters (described in JP-A-2002-320477); GTP binding protein promoter, ATP synthase promoter, chaperone promoter, heat shock protein promoter, heat shock protein activation protein promoter, G4 nucleic acid binding protein promoter, ATP / ADP carrier Protein promoter (described in JP-A-2003-61659); Like Aspergillus origin of manganese superoxide dismutase promoter (described in JP 2003-164283) and the like. Among these, sodM promoter, melO promoter, and glaA promoter are preferable, and sodM promoter is more preferable.
<Substrate compound>
As the substrate compound, at least one compound selected from the group consisting of tyrosine, DOPA and DOPA analogs is used. Tyrosine, DOPA and DOPA analogs may be either L-form or D-form. Analogs include dopamine, DOPA or tyrosine lower (1 to 4 carbon) alkyl esters, and α-lower (1 to 4 carbon) alkyl DOPA, α-lower (1 to 4 carbons). Examples thereof include alkyltyrosine. Among them, L-tyrosine and / or L-DOPA is preferably used from the viewpoint of obtaining a natural melanin precursor, and L-DOPA is more preferably used from the viewpoint of affinity for the enzyme.

A substrate can be used individually by 1 type or in combination of 2 or more types.
<Reaction>
The substrate concentration at the start of the reaction is usually preferably about 10 to 60 mM, more preferably about 15 to 25 mM. If it is the said range, while a sufficient amount of melanin can be obtained, the yield reduction of the melanin precursor by the excessive amount of melanin production | generation and the residual of unreacted DOPA will not arise.

  The pH of the reaction solution is not particularly limited as long as the enzyme can catalyze the oxidation reaction of the substrate, but is usually preferably maintained at about 4 to 9, more preferably about 5 to 7. If the pH is too low, the oxidation of the substrate will not proceed. Conversely, if the pH is too high, the produced melanin will polymerize without accumulating. The melanin precursor can be efficiently accumulated therein.

The pH of the reaction solution can be maintained in the above range by using a buffer solution as the reaction solution, but if the salt concentration is high, the production of melanin by polymerization of the melanin precursor may be promoted, so KOH, It is preferable to adjust by adding a small amount of a strong alkali such as NaOH and a strong acid such as H ₂ SO ₄ and HCl.

The amount of cells in the reaction solution is preferably as small as possible so that the catechol oxidase activity is 5 × 10 ⁵ U / mol or more when 1 mol of L-DOPA is used as a substrate. The cell input amount is usually preferably 20% by volume or less, more preferably 10% by volume or less, based on the volume of the reaction solution. If it is in said range, the yield of a melanin precursor can be made high because the bacterial cell separation after reaction can be performed easily.

  The reaction temperature is not particularly limited as long as the enzyme can catalyze the oxidation reaction of the substrate, but is usually preferably maintained at about 15 to 35 ° C, more preferably about 20 to 30 ° C. Within the above range, the oxidation reaction proceeds sufficiently, the enzyme is hardly deactivated, and melanization is difficult to proceed.

  Immediately after the start of the reaction, a large amount of oxygen is required for the oxidation reaction. However, since the cells are damaged when the stirring speed is too high, it is preferable to monitor the oxygen concentration in the reaction solution and to reduce the aeration amount and stirring speed if the oxygen concentration does not decrease. The oxygen concentration in the reaction solution is preferably maintained at about 0.1 to 8 ppm, more preferably about 1 to 2 ppm. When a large amount of foam is generated in the reaction solution by aeration or stirring, an antifoaming agent such as a silicone resin may be added.

  The reaction may be either batch type or continuous type. The batch method is preferable in that the unreacted substrate and the product can be separated. In the case of a batch system, the reaction time is usually preferably about 10 minutes to 2 hours, more preferably about 30 minutes to 1 hour. If the reaction is carried out for too long, the polymerization reaction of the produced melanin precursor will proceed and the yield will decrease, but at this level, the substrate compound will be sufficiently converted to the melanin precursor while minimizing the polymerization reaction. it can.

  In the case of the continuous method, the reaction solution may be continuously collected while supplying the substrate to the reaction vessel containing cells so that the substrate concentration is about 10 to 60 mM, particularly about 15 to 25 mM. Alternatively, a column packed with a carrier on which an enzyme or cells are immobilized may be added with a substrate of about 1 to 10 mM, particularly about 3 to 6 mM, and hydrogen peroxide twice the concentration of the substrate as an electron donor. .

  When tyrosine or DOPA is used as the substrate compound, the substrate compound is oxidized by the above enzyme reaction to produce dopachrome. If this reaction solution is allowed to stand, 5,6-dihydroxyindole is produced by spontaneous decarboxylation of dopachrome, or it is produced from dopachrome by dopachrome tomerase contained in cells or by non-enzymatic isomerization. 5,6-Dihydroxyindole-2-carboxylic acid is produced. This gives a melanin precursor containing dopachrome, 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid.

  After the oxidation step using cells, the melanin precursor having a high dopachrome ratio can be obtained by immediately moving to the recovery step and adjusting the composition by blocking the supply of oxygen to the reaction solution.

  Further, when dopamine, for example, is used as the substrate compound, dopamine chrome is generated by oxidation of the substrate compound. If this reaction solution is allowed to stand, dihydroxyindole is produced, and a melanin precursor can also be obtained.

  The melanin precursor may contain a water-soluble oligomer obtained by polymerizing 2 to 5 molecules thereof.

  In addition, by adding a thiol compound such as cysteine or glutathione or a heterogeneous compound such as kojic acid to the substrate, these heterogeneous compounds bind to the melanin precursor constituents described above, and / or A melanin precursor in which different compounds are mixed is obtained. These heterogeneous compounds can also be added to the reaction solution during or after the oxidation reaction step, or during or after the composition adjustment step described later. Modified melanin precursors obtained by using heterogeneous compounds that do not fall under any of tyrosine, DOPA or DOPA analogs are also included in the melanin precursor of the present invention.

The melanin precursor obtained by using DOPA or dopamine as a substrate becomes black melanin by polymerization. Moreover, the melanin precursor obtained by using the alkyl ester of tyrosine or the alkyl ester of DOPA as a substrate becomes yellow melanin by polymerization. Melanin precursor obtained by using α-alkyl ester of tyrosine or α-alkyl ester of DOPA as a substrate becomes black melanin by polymerization, but yellow to purple melanin is synthesized by adjusting pH and polymerizing can do. The melanin precursor obtained by using tyrosine or DOPA and cysteine together as a substrate becomes brown melanin by polymerization.
Step of adjusting composition As described above, when the reaction solution is allowed to stand after completion of the oxidation step, melanin is generated without generating 5,6-dihydroxyindole or 5,6-dihydroxyindole-2-carboxylic acid from dopachrome. On the other hand, the composition of the melanin precursor can be adjusted by performing the following treatment after removing the cells from the reaction solution by a method such as centrifugation after the oxidation step. After the oxidation reaction is completed, it is desirable that the reaction solution is in a state of blocking oxygen in order to prevent oxidation.
(i) Production of a precursor solution having a high ratio of 5,6-dihydroxyindole-2-carboxylic acid
<Composition adjustment process (A)>
After completion of the oxidation step with catechol oxidase, about 0.1 to 20 mM, preferably about 5 to 10 mM of divalent copper ion is added to the reaction solution, and stored for about 10 to 30 minutes, for example, so that dopachrome in the melanin precursor is stored. Is promoted to 5,6-dihydroxyindole-2-carboxylic acid.

  The temperature at the time of a contact with a melanin precursor and a bivalent copper ion is not specifically limited, What is necessary is just to perform at about 20-30 degreeC same as an oxidation process. The reaction solution may be allowed to stand at room temperature.

  In the present invention, “preservation” is not a term indicating only standing in a fixed state, but includes a case where temperature, treatment agent concentration, and the like change. Moreover, the case where it shakes other than the case where it leaves still is included.

  Copper ions can be added, for example, as a copper sulfate solution.

One of the factors that determine the ease of dyeing is the electrical properties of the dye. When the melanin precursor obtained by the method of the present invention is used as a dye, the electrical properties of the dye differ depending on the ratio of 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid in the melanin precursor . Therefore, it is convenient if the ratio of 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid can be appropriately adjusted depending on the material to be dyed. Melanin precursors rich in 5,6-dihydroxyindole-2-carboxylic acid are suitable for dyeing fibers made of keratin, for example.
<Composition adjustment process (B)>
Further, after the oxidation step with catechol oxidase, the melanin precursor as a reaction product can be stored in the presence of a buffer, usually at about -80 to 0 ° C, preferably at about -30 to -10 ° C. This also promotes the conversion of dopachrome in the melanin precursor to 5,6-dihydroxyindole-2-carboxylic acid.

  Specifically, a buffer solution may be obtained by adding a buffer to the reaction solution. The type of buffer is not particularly limited as long as it has a buffer capacity in the range of about pH 4 to 9, but it is phosphorous in that the conversion efficiency to 5,6-dihydroxyindole-2-carboxylic acid is increased. Acid buffer is preferred. The phosphate buffer may be a buffer containing only phosphoric acid or may contain other components. Examples of the former include sodium phosphate buffer and potassium phosphate buffer. Examples of the latter include citrate-sodium phosphate buffer and tris-phosphate buffer.

  The concentration of the buffer in the reaction solution may be, for example, about 10 mM to 500 mM, preferably about 50 mM to 200 mM. If it is said buffering agent concentration range, while the effect of a buffering agent will fully be acquired, superposition | polymerization of melanin will not be accelerated | stimulated.

  Moreover, if it is said storage temperature range, while conversion reaction will advance sufficiently efficiently, the production amount of melanin will not increase easily.

The storage period is usually about 1 to 7 days.
(ii) Production of a precursor solution having a high ratio of 5,6-dihydroxyindole
<Composition adjustment process (C)>
Moreover, the melanin precursor which is a reaction product can be preserve | saved in presence of a salt after the oxidation process by catechol oxidase. This promotes the conversion of dopachrome in the melanin precursor to 5,6-dihydroxyindole.

  The kind of salt is not specifically limited, A well-known salt can be used without a restriction | limiting. Examples of the salt include alkali metal salts and alkaline earth metal salts of acids such as hydrochloric acid, nitric acid, sulfuric acid, carbonic acid, acetic acid, and phosphoric acid.

  The salt concentration in the reaction solution may be, for example, about 0.5 to 40% by weight, preferably about 1 to 5% by weight. If the salt concentration is too high, salt or melanin may precipitate, but if it is within the above range, conversion to 5,6-dihydroxyindole is efficiently performed, and salt or melanin does not precipitate.

The storage temperature is not particularly limited, and may be about 20 to 30 ° C., which is the same as that in the oxidation step. The reaction solution may be allowed to stand at room temperature. The storage period is usually about 30 minutes to 3 hours.
<Composition adjustment process (D)>
After the oxidation step with catechol oxidase, the pH of the reaction solution is adjusted to about 5 to 7, and the reaction solution is stored in the presence of oxygen, that is, in the atmospheric air, so that 5,6- dopachrome contained in the melanin precursor is stored. Conversion to dihydroxyindole is facilitated.

  Also, the conversion to 5,6-dihydroxyindole can be promoted by adjusting the pH of the reaction solution to about 5 to 10 and storing the reaction solution in a state where oxygen is not present or almost absent. . 5,6-dihydroxyindole is produced at a pH of about 5 to 10, but the produced 5,6-dihydroxyindole is rapidly oxidized at a pH of about 7 to 10, so that the efficiency is improved by working in the absence of oxygen. Thus, 5,6-dihydroxyindole can be obtained.

  Furthermore, the pH of the reaction solution is adjusted to 5 or less, and the precipitate obtained by storing the reaction solution in a state where oxygen is not present or almost absent is at a pH of 5 to 7, in a state where oxygen is absent or almost absent. The 5,6-dihydroxyindole is obtained by dissolving in

  The state where oxygen is not present or almost absent can be achieved by sealing the reaction vessel or replacing the upper gas of the reaction vessel with nitrogen gas, inert gas (rare gas) or carbon dioxide gas.

The storage temperature is not particularly limited, but may be stored at about 5 to 40 ° C., for example. It is sufficient to store for 3 to 24 hours.
<Composition adjustment process (E)>
In addition, the ratio of 5,6-dihydroxyindole in the melanin precursor can also be improved by adding a neutral or acidic water-soluble organic solvent to the reaction solution after the oxidation step with catechol oxidase. .

  Examples of such water-soluble organic solvents include lower alcohols such as methanol, ethanol, propyl alcohol, isopropyl alcohol, butyl alcohol, isobutyl alcohol, s-butyl alcohol, and t-butyl alcohol; lower alkyl esters such as acetic acid ethyl ester; Examples include ethyl ether, acetone, acetic acid, acetonitrile, and the like. In particular, alcohol is preferable from the viewpoint of safety, and ethanol and acetone are more preferable.

  The addition amount of the aqueous organic solvent may be such that the ratio of alcohol to the total amount of the reaction solution is usually about 10 to 70% by volume, preferably about 40 to 60% by volume. Even if the amount of the solvent added is increased, there is no particular problem. On the contrary, the conversion rate is increased, and the produced melanin can be removed. The ratio of dihydroxyindole can be improved and aftertreatment can be performed safely.

  The storage temperature is not particularly limited, but may be, for example, about 5 to 40 ° C. In addition, it is sufficient to store for about 30 minutes to 1 hour.

The aforementioned salt treatment, deoxygenation treatment under pH 5-7, and neutral or acidic water-soluble organic solvent treatment can be performed in combination of two or more. The implementation order in this case is not particularly limited. Moreover, a salt and / or a water-soluble organic solvent can be added to the reaction solution, or / and the pH can be adjusted to 5 to 7 and stored in the absence of oxygen. As a result, the ratio of 5,6-dihydroxyindole can be further improved.
Step of recovering melanin precursor After completion of each of the above steps, the melanin precursor is recovered. The melanin precursor can be recovered by removing cells in the reaction solution by means such as centrifugation. The removed cells can be reused by returning them to the container for the oxidation step. In addition, cell removal is unnecessary when the cell has already been removed before the composition adjustment step.

  Moreover, you may refine | purify a melanin precursor from a reaction liquid. That is, the reaction solution after completion of the reaction may contain a protein produced by damaging cells by aeration and stirring or a protein flowing out from the cells. Therefore, it is preferable to perform protein removal by a known method such as ultrafiltration or gel filtration chromatography. When yeast cells are used as cells for producing catechol oxidase, the cell wall is strong, so there are few or almost no cell-derived proteins present in the reaction solution, but melanin precursors are used as pharmaceuticals and foods. In this case, since it is desirable to strictly remove such cell-derived protein, it is preferable to perform a protein removal step.

  Further, the reaction liquid usually contains melanin obtained by polymerization of these in addition to the melanin precursor. Therefore, it is preferable to remove melanin by a method such as ultrafiltration.

  Further, it is preferable to concentrate the melanin precursor by removing moisture by a known method such as reverse osmosis concentration, spray drying, freeze concentration and the like.

  The reverse osmosis concentrator is used for producing pure water from seawater, and since it can process a large amount of solution, it is suitable for industrially concentrating a melanin solution. There are two types of reverse osmosis concentrators, a batch type and a cross flow type, but when oxygen is present, the chemical oxidation of the precursor gradually proceeds, so air does not enter the container when using the batch type. It is desirable to devise as follows. Also, it is better to use nitrogen as a pressure source with as little oxygen content as possible. The cross-flow type apparatus is usually pressurized by a pump, but it is necessary to circulate the non-permeated liquid. Therefore, it is desirable to replace the melanin precursor solution with high-purity nitrogen in advance and to make the liquid receiving tank sealed.

In the recovery step, it is preferable to shut off oxygen by placing the melanin precursor solution in a sealed container or further replacing the upper gas with nitrogen, rare gas, carbon dioxide gas or the like. Thereby, progress of the oxidation of a melanin precursor can be suppressed and it can be set as the melanin precursor of a desired composition. Preservation of melanin precursor A single product or a mixture of melanin precursors can be preserved in any form such as a concentrated state or a dry powder state.

Dopachrome can be stably stored by freezing at a low temperature of -40 ° C or lower. In addition, 5,6-dihydroxyindole can be stably stored by freezing in the absence of oxygen or at a low temperature of -40 ° C or lower, and 5,6-dihydroxyindole-2-carboxylic acid can be stored in the absence of oxygen or at 0 ° C. It can be stably stored by freezing at the following low temperatures. Except for dopachrome, it is highly practical because it can be stored at room temperature if oxygen is removed.
(I) Method for producing second melanin precursor
Basic Structure The second method for producing a melanin precursor of the present invention comprises 1 mol of an enzyme having catechol oxidase activity for at least one substrate compound selected from the group consisting of tyrosine, DOPA and analogs thereof. An oxidation step of oxidizing the substrate compound to convert it into a melanin precursor by reacting with catechol oxidase activity of 5 × 10 ⁵ U / mol or more when using L-DOPA as a substrate; reaction A recovery step of recovering the melanin precursor from the liquid.
Oxidation process of substrate compound
<Enzyme having catechol oxidase activity>
The type of enzyme having catechol oxidase activity is the same as in the first production method. The origin of the enzyme is not particularly limited, and it may be derived from any organism such as bacteria, fungi, plants, and animals.
This enzyme may be used as it is added to the reaction solution. Further, by using an immobilized enzyme, the stability of the enzyme in the reaction solution is improved, and it can be easily separated from the reaction system after use. There is also an advantage that no protein is mixed into the reaction system. The method for immobilizing the enzyme is not particularly limited, and a known method can be adopted. Examples of known immobilization methods include a method of crosslinking enzyme molecules with an immobilization carrier, a method of encapsulating in a gel such as an alginate gel, and the like.
The enzyme may be a crude sample containing contaminants derived from living organisms or a purified enzyme, but it is preferably purified when immobilized.
In the present invention, an excessive amount of enzyme is allowed to act on the substrate. The activity when 1 mol of L-DOPA is used as a substrate is usually 5 × 10 ⁵ U / mol or more, preferably 5 × 10 ³ U / mol or more. The upper limit of the catechol oxidase activity per 1 mol of L-DOPA is not particularly limited, but usually about 5 × 10 ⁷ U / mol is sufficient.

Similarly to the first production method, it is desirable to activate an enzyme having catechol oxidase activity by coordination of divalent copper ions or treatment with an acidic solution having a pH of about 2.8 to 3.2. The enzyme can also be activated by treatment with a protease such as endopeptidase that selectively cleaves specific peptide bonds such as trypsin. By treating with a protease, the N-terminal and / or C-terminal sequences of the enzyme are removed, and the enzyme activity is improved.
<Substrate compound>
The substrate compound is the same as in the first production method.
<Reaction>
The method for adjusting the oxidation reaction of the substrate and the composition of the melanin precursor is the same as in the first production method.
Step of recovering melanin precursor After completion of each of the above steps, the melanin precursor is recovered. The melanin precursor can be recovered by removing the enzyme in the reaction solution by means such as ultrafiltration, filtration, and centrifugation. The removed enzyme can be reused by returning it to the container for the oxidation step. In addition, enzyme removal is unnecessary when the enzyme has already been removed before the composition adjustment step.
Storage of melanin precursor The storage method of the melanin precursor is the same as in the case of the first production method.
EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
<Quantification method of each component of melanin precursor>
In each Example, each component of the melanin precursor was detected and quantified under the following conditions using Waters HPLC Alliance2695-2996.

  Separation of components using Imtakt reverse phase column Unison UK-C18 (4.6 x 150 mm), 1.5% phosphoric acid solution (A solution) and 99.9% methanol (B solution) as the mobile phase. The gradient was set so that the B liquid was 0% at the initial stage and 50% after 5 minutes. The flow rate was 1.0 ml / min.

For the sample to be injected, add 10 μl of sample to 100 μl of 20 mM sodium dithionite (Na ₂ S ₂ O ₄ ) and 890 μl of 1.5% phosphoric acid (H ₃ PO ₄ ) solution, and use a 0.45 μm filter. Prepared by filtration. 20 μl of this was injected into the above column for measurement.

  The detection of dopa was monitored by the absorbance at 280 nm, which is the maximum absorption wavelength. Dopachrome is quantified as reduced leucodopachrome under the above measurement conditions. 5,6-dihydroxyindole can be quantified because of the presence of a standard substance, but 5,6-dihydroxyindole-2-carboxylic acid cannot be quantified because there is no standard substance. Therefore, each component concentration was simply compared with the peak area at 280 nm. However, since the maximum absorption wavelength of 5,6-dihydroxyindole is 300 nm and the maximum absorption wavelength of 5,6-dihydroxyindolecarboxylic acid is 320 nm, 5,6-dihydroxyindole and 5,6-dihydroxyindole- 2-Carboxylic acid has a peak area that is less than the actual concentration.

(Cloning of Aspergillus oryzae tyrosinase gene)
As a result of intensive studies, the inventors have found that Aspergillus oryzae expresses a large amount of melB tyrosinase gene in solid culture such as rice bran (Japanese Patent Laid-Open No. 2002-191366). Based on the analyzed genomic base sequence of melB gene, reverse transcription PCR was performed by a conventional method to clone cDNA.

Specifically, the Aspergillus oryzae OSI-1013 strain (deposited as FERM P-16528 at the National Institute of Advanced Industrial Science and Technology as the FERM P-16528) was inoculated into steamed rice, and 1.5 g of the koji produced in a conventional manner was weighed, and the liquid Completely crushed in nitrogen. Subsequently, 240 μg of total RNA was extracted using ISOGEN ^™ manufactured by Nippon Gene Co., Ltd. as described in the instruction manual. Furthermore, 1 μg of mRNA was purified from 120 μg of total RNA using Oligotex ^™ -dT30 <Super> manufactured by Takara Bio Inc. according to the description in the instruction manual. Based on the mRNA thus obtained, a cDNA library was prepared by SMART ^™ cDNA Library Construction Kit manufactured by Clontech, and only melB cDNA was amplified by PCR.
To describe the reaction conditions in detail, 1 μl each of SMART III oligo solution and CSD III / 3 ′ PCR primer solution attached to the kit was added to 3 μl mRNA solution (including 50 ng mRNA) and kept at 72 ° C. for 2 minutes. Then, it was rapidly cooled on ice for 2 minutes. After centrifugation, 2 μl of 5 × First Strand buffer, 1 μl of DTT (20 mM) solution, 1 μl of dNTP Mix (10 mM) solution, and 1 μl of MMLV RTase solution were added, and reverse transcription reaction was performed at 42 ° C. for 1 hour. The obtained cDNA library was subsequently used as a template for the specific amplification of melB cDNA. 2 μl cDNA library solution, 80 μl sterile distilled water, 10 μl 10 × Advantage 2 PCR buffer, 2 μl 50 × dNTP mix solution, 2 μl 5 ′ PCR primer (sequence from melB start codon; 5′-atgccctacc tcatcaccgg tatcccaaag -3 ′; SEQ ID NO: 1) solution, 2 μl of CSD III / 3 ′ PCR primer solution, and 2 μl of 50 × Advantage 2 polymerase mix solution were added. The reaction was completed by using 30 cycles of 95 ° C for 5 seconds and 68 ° C for 6 minutes using PerkinElmer's thermal cycler PE2400.
The obtained PCR product was subjected to agarose gel electrophoresis, and it was confirmed that only the target band of about 1.8 Kbp was amplified. As a result of the base sequence analysis, it was also confirmed that the intron sequence was normally removed.

Integration into yeast According to Example 1, cDNA was expressed as an expression vector pET23b for Escherichia coli manufactured by Invitrogen, a yeast Saccharomyces cerevisiae expression vector (using SED1 promoter) described in Japanese Patent JP-A 2003-265177, and Japanese Patent JP By connecting to the Aspergillus oryzae expression vector described in 2001-224381 (using sodM promoter) in an expressible state, large-scale expression in each cell was successful. Only the expression in yeast is described in detail here.
As a result of the inventors' diligent research, the SED1 promoter was found to be very strongly expressed from the latter half of the logarithmic growth phase.

  The plasmid construction method is shown in FIG. The melB cDNA amplified by PCR was inserted into the SmaI site immediately below the promoter of the expression vector having the SED1 promoter and ADH1 terminator. Furthermore, a fragment containing melB cDNA obtained by cutting at the StuI site present inside the URA3 marker was purified as an introduction cassette.

  The yeast used as the host was a uracil auxotrophic strain derived from the Practical Yeast Association No. 9 used for sake brewing. The reason why the practical yeast is used instead of the laboratory yeast is that the diploid is used, so that two copies of the vector may be introduced. It is known that there are triploid and tetraploid yeasts in practical use. If these yeasts are used, transformants having a larger copy number can be obtained.

Measurement of tyrosinase activity in recombinant yeast
<Activation treatment of tyrosinase>
The recombinant yeast obtained in Example 2 collected the cells by centrifugation, and then washed with distilled water to remove the medium components well. The cells were suspended in 50 mM Tris-HCl buffer (pH 8.0) containing 2 mM copper sulfate, which was about 10 times the mass of the cells, and allowed to stand at 4 ° C. overnight. The cells were collected by centrifugation and washed with 0.1 M EDTA solution to remove excess copper ions.

Following the copper addition treatment, an acid activation treatment was performed. That is, the cells after copper addition treatment and washing were suspended in 0.2 M acetate buffer (pH 3.0) and allowed to stand at room temperature for 1 hour. The activated cells were collected by centrifugation.
<Measurement of tyrosinase activity>
The tyrosinase activity of the bacterial cells was measured as follows. That is, a part of the collected cells were suspended in water, 0.8 ml of 10 mM L-DOPA (dissolved in 0.005N hydrochloric acid) kept at 30 ° C. with respect to 0.1 ml and 1 M sodium phosphate After adding 0.1 ml of buffer solution (pH 6.0) and reacting at 30 ° C. for 5 minutes, the cells were removed by centrifuging at 15,000 rpm for 30 seconds, and the absorbance at 475 nm, which is the maximum absorption wavelength of DOPA. Was measured. The amount of suspended cells was adjusted so that the absorbance at 475 nm of the reaction solution after the reaction was 0.1 to 0.3 for the convenience of the experiment.
In the present invention, 1U of tyrosinase activity is defined as an activity that increases the absorbance at 475 nm when a 1 ml DOPA solution is reacted at 30 ° C. for 5 minutes, and is divided by the density of the cells used in the reaction (absorbance at 600 nm). This was regarded as the tyrosinase activity of the bacterial cells.

Tyrosinase activity of bacterial cells = U / OD ₆₀₀
The yeast having the highest tyrosinase activity (tyrosinase activity: 1.9 to 4.4 U / OD ₆₀₀ ) among the transformed strains thus measured was cultured to the stationary phase using YPD medium, and used in Examples 4 and below. .

Accumulation reaction of dopachrome Using the yeast transformant obtained in Example 3, an accumulation reaction of dopachrome, which is a melanin precursor, was performed using L-DOPA as a substrate. The same reaction can be performed with recombinant Escherichia coli and recombinant rod-shaped cells, but here, the reaction using yeast is described in particular.

As described above, the initial concentration of L-DOPA can be increased to obtain as high a melanin precursor solution as possible, but here the residual DOPA is below the detection limit and a high concentration of melanin precursor is obtained. The method is described.
When performing reaction of 2L reaction solution in 5L reaction tank, dissolve 7.9g of L-DOPA in 500ml of 0.03NH ₂ SO ₄ and use 2N KOH to adjust the pH of this solution to the optimum pH of melB tyrosinase. The pH was adjusted to 5.5. Recombinant yeast cells (obtained in Example 3) with an activity value of 1.35 × 10 ⁵ U / L per liter of reaction solution are added to this L-DOPA solution in a batch reaction tank. The reaction was started.
The temperature was adjusted with cold water and a heater so that the reaction temperature would be 25 ° C., the optimum temperature.
Immediately after the start of the reaction, a large amount of oxygen is required as an acceptor for hydrogen ions generated by the oxidation reaction, so the aeration amount and the stirring speed were maximized. In addition, since the pH dropped at the beginning of the reaction, 2N KOH was added dropwise to adjust the pH to 5.5. Dopachrome is chemically oxidized. At that time, since the pH increased, 0.3N H ₂ SO ₄ was added dropwise to adjust the pH to 5.5. The pH was adjusted automatically by an online monitor.
In this way, the reaction was carried out for 40 minutes.
The transition of the aeration amount and the stirring speed in the reaction solution is shown in FIG. Moreover, transition of pH and oxygen concentration (OD) of the reaction solution is shown in FIG. Further, FIG. 5 shows the cumulative amount of 2N KOH solution and 0.3NH ₂ SO ₄ solution added to maintain the pH of the reaction solution at 5.5.
Observation of reaction progress Sampling was appropriately performed during the reaction and rapidly frozen at -80C and stored. After the reaction, the stored sample was quantified for dopachrome and 5,6-dihydroxyindole by the method described above. The transition of the L-DOPA concentration and the melanin precursor concentration in the reaction solution is shown in FIG.
Under these reaction conditions, 5,6-dihydroxyindolecarboxylic acid is hardly formed and is negligible, so the precursor concentration obtained is the sum of the dopachrome concentration and the 5,6-dihydroxyindole concentration.
In addition, the time-dependent change of the melanin precursor density | concentration was collated with the time-dependent change of the acid and alkali addition amount for recording oxygen concentration and pH maintenance, etc., and the criterion for judging the completion of the reaction was set. In order to bring L-DOPA below the detection limit, the reaction was carried out longer than previously determined conditions.

Deproteinization of the precursor solution Since the melanin precursor solution obtained in Example 4 uses yeast cells having a strong cell wall, the fragments of the cells present in the melanin precursor solution and the proteins that have flowed out of the cells Is considered to be absent or almost absent. However, when this product is used as a food or medicine, it is desirable that no protein is contained, and therefore protein removal was performed by ultrafiltration.

  As an ultrafiltration device, an ultrafiltration module Molsep FS10-FUS0181 (fractional molecular weight 10,000) manufactured by Daisen Membrane Co. was used. The solution obtained by filtration was subjected to SDS-PAGE, and silver staining was performed to confirm that the protein was deproteinized. The method of measuring the absorbance at 280 nm as is generally done was not adopted because dopachrome itself has absorption near 280 nm, and the indole has high reactivity, so it easily reacts with the color reagent. Because of the reason. As a result of silver staining, no band considered to be protein was detected after ultrafiltration.

Concentration of precursor solution In order to increase the concentration of melanin precursor obtained by the reaction, if the initial substrate (L-DOPA) concentration is increased, L-DOPA is contained in the melanin precursor solution obtained after the reaction. Detected. Conversely, when a relatively low concentration of L-DOPA as used in Example 4 is used, a high concentration melanin precursor solution cannot be obtained.
Therefore, the melanin precursor solution deproteinized in Example 5 was concentrated as follows. For the concentration, a cross flow type reverse osmosis concentration device (manufactured by Nitto Denko Matex Co., Ltd.) used for producing pure water from seawater was used. As shown in FIG. 7, this apparatus circulates a solution between a closed tank containing a melanin precursor solution and a reverse osmosis concentration module NTR7410-HG-S4F. The pure water to be generated is led from this module to the permeate tank. As pure water is produced, the melanin precursor is concentrated in the tank. Circulation was performed by a pump at a pressure of 2 Mpa.
As a result, 50 L of melanin precursor solution was concentrated to about 30 L by operation for about 30 minutes. The solution during concentration was sampled over time, and the concentration of 5,6-dihydroindole in the melanin precursor was measured by HPLC. Concentration produced almost no melanin in the melanin precursor permeate, and the recovery rate of the melanin precursor (represented by the 5,6-dihydroindole concentration) was 85% or more. The transition of the 5,6-dihydroindole concentration in the solution by concentration is shown in FIG.
The concentrated solution thus obtained could be stored frozen at -80 ° C. Further, when the concentrated solution was replaced with an ultrapure nitrogen gas and then sealed in an ampoule and stored at 40 ° C., it could be stably stored for one year as shown in FIG.

Powdered <br/> producing melanin precursor The precursor may be powdered. The melanin precursor solution obtained in Example 6 was pulverized using Spray Dry Parvis GB22 manufactured by Yamato Kagaku.

  The concentrated melanin precursor obtained in Example 6, the concentrated melanin precursor encapsulating the oxygen scavenger, the dried melanin precursor, and the dry melanin precursor encapsulating the oxygen scavenger were each stored at 40 ° C. The results of measuring the 5,6-dihydroxyindole content over time are shown in FIG. As is clear from FIG. 10, it can be seen that the melanin precursor can be stably stored for one year by enclosing the oxygen scavenger and leaving it in the absence of oxygen. In particular, high stability was obtained by blocking oxygen in the dry state.

  Although not shown, the dried melanin precursor enclosed with an oxygen scavenger could be stably stored at 40 ° C. for 3 months or more.

Control of precursor solution composition by pH Five samples were prepared for the melanin precursor solution deproteinized according to Example 5 (mainly containing dopachrome) and the pH was 2, 4, 6, 8 and 10 respectively. And all samples were sealed in microtubes so that no oxygen was present in the container and stored at 40 ° C. for 2 hours.
The 5,6-dihydroxyindole-2-carboxylic acid concentration and the 5,6-dihydroxyindole concentration contained in each sample after storage were measured using HPLC.

  The results are shown in FIG. As a result, when stored at pH 5-7, the ratio of 5,6-dihydroxyindole is increased, and it is understood that almost all dopachrome is converted to 5,6-dihydroxyindole. The higher the pH, the greater the proportion of 5,6-dihydroxyindole-2-carboxylic acid in the melanin precursor solution. In addition, precipitation occurred at pH 2 and pH 4.

Composition control of precursor solution by organic solvent Ethanol is added to 50% by volume of the total amount of melanin precursor solution (mainly containing dopachrome) obtained by deproteinization in Example 5. And stored at room temperature for 2 hours. Similarly, acetone and water were added respectively so that it might become 50 volume% with respect to the whole quantity of a precursor solution, and it preserve | saved at room temperature for 2 hours. The concentrations of dopachrome, 5,6-dihydroxyindole-2-carboxylic acid and 5,6-dihydroxyindole in each solution after storage were measured by HPLC.

The results are shown in FIG. By storing the melanin precursor solution in the presence of neutral or acidic water-soluble solvents methanol, ethanol, acetone, acetone, 2-propanol and acetonitrile, dopachrome is efficiently converted to 5,6-dihydroxyindole You can see that In addition, by storing the melanin precursor solution in the presence of 2-aminoethanol, a basic water-soluble organic solvent, dopachrome was efficiently converted to 5,6-dihydroxyindole-2-carboxylic acid. I understand.
In addition, it can be seen that by adding ethanol or acetone for storage, the yield of 5,6-dihydroxyindole increases as compared with the case of storing by adding the same amount of water.

Control of composition of precursor solution by phosphate buffer By adding 1M phosphate buffer to the melanin precursor solution (mainly containing dopachrome) obtained by deproteinization in Example 5. A pH 6.0 0.1M phosphate buffer containing a melanin precursor was prepared. This precursor solution was stored at about −20 ° C. for 7 days, sampled over time, and dopachrome, 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid were quantified by HPLC.
The results are shown in FIG. FIG. 13 shows that dopachrome was efficiently converted to 5,6-dihydroxyindole-2-carboxylic acid by low-temperature storage in a phosphate buffer containing phosphoric acid as an acid component. As mentioned above, since the concentration calculated from the peak area of 280 nm of 5,6-dihydroxyindole-2-carboxylic acid is lower than the actual concentration, in reality, almost all of dopachrome is 5,6-dihydroxyindole. It is thought that it has been converted to -2-carboxylic acid.

Control of composition of precursor solution by salt solution Sodium chloride and ascorbine were added to the melanin precursor solution (mainly containing dopachrome) obtained by deproteinization in Example 5 to a final concentration of 2.5%. Each of the sodium acid solutions was added and allowed to stand at room temperature for 45 minutes.

The concentrations of dopachrome, 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid in the melanin precursor solution after standing were measured by HPLC. Moreover, it measured similarly about the control solution which left the melanin precursor solution as it was at room temperature, without adding salt.
The results are shown in FIG. As is apparent from FIG. 14, it can be seen that the addition of salt to the melanin precursor promoted the conversion of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid. In particular, when sodium ascorbate is added, the antioxidant action has a synergistic effect, and it can be seen that a significant amount of 5,6-dihydroxyindole accumulates.

Composition control of precursor solution by divalent copper ions Copper sulfate was added to the melanin precursor solution (mainly containing dopachrome) obtained by deproteinization in Example 5 so that the final concentration was 1 mM, and at room temperature. Let stand for 15 minutes.

  The concentrations of dopachrome, 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid in the melanin precursor solution after standing were measured by HPLC. Moreover, it measured similarly about the control solution which left the melanin precursor solution as it was at room temperature, without adding a bivalent copper ion.

  The results are shown in FIG. As is clear from FIG. 15, it was found that the conversion of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid was promoted by contacting the melanin precursor with a divalent copper ion.

It is a figure which shows the biosynthesis pathway of melanin. It is a figure explaining the construction method of the melB tyrosinase gene introduction cassette of Aspergillus oryzae. In Example 4, it is a figure which shows transition of the stirring speed and ventilation | gas_flowing amount in the oxidation reaction of L-DOPA by the tyrosinase production cell. In Example 4, it is a figure which shows transition of pH and oxygen concentration of the reaction liquid in the oxidation reaction of L-DOPA by the tyrosinase producing cell. In Example 4, it is a figure which shows the cumulative amount of the acid and alkali added during the oxidation reaction of L-DOPA by the tyrosinase producing cell. In Example 4, it is a figure which shows transition of the L-DOPA density | concentration and the melanin precursor density | concentration in the reaction liquid of the oxidation reaction of L-DOPA by the tyrosinase producing cell. It is a figure which shows schematic structure of the reverse osmosis concentration apparatus used for the concentration of the melanin precursor solution in Example 6. It is a figure which shows transition of the 5, 6- dihydroxyindole density | concentration in the concentration process of the melanin precursor solution of Example 6, and a concentration rate. It is a figure which shows that the concentrated melanin precursor was preserve | saved at 40 degreeC in oxygen absence. It is a figure which shows that the dried melanin precursor was able to preserve | save at 40 degreeC in oxygen absence. It is a figure which shows the adjustment result of the composition of the melanin precursor solution by pH. It is a figure which shows the adjustment result of the composition of the melanin precursor solution by a water-soluble organic solvent. It is a figure which shows the adjustment result of the composition of the melanin precursor solution by a phosphate buffer. It is a figure which shows the adjustment result of the composition of the precursor solution by a salt solution. It is a figure which shows the adjustment result of the composition of the melanin precursor solution by bivalent copper ion.

Claims (20)

In the reaction solution, at least one substrate compound selected from the group consisting of tyrosine, DOPA and analogs thereof, and one or more factors of (a), (b), (c) and (d) below: An oxidation step of oxidizing a substrate compound and converting it into a melanin precursor in the presence of cells exhibiting high catechol oxidase activity in
The manufacturing method of a melanin precursor including the collection | recovery process which collect | recovers a melanin precursor from a reaction liquid.
(a) This gene is expressed under a promoter having a higher activity than a promoter that originally expresses a gene encoding a polypeptide having catechol oxidase activity.
(b) It has multiple copies of a gene encoding a polypeptide having catechol oxidase activity.
(c) High catechol oxidase activity is exhibited by having a mutated gene encoding a polypeptide having catechol oxidase activity.
(d) High catechol oxidase activity is exhibited by tyrosinase activation treatment. The method according to claim 1, wherein the catechol oxidase activity of the cell when L-DOPA is used as a substrate is 0.1 U / OD ₆₀₀ or more. The method according to claim 1 or 2, wherein the catechol oxidase activity when 1 mol of L-DOPA is used as a substrate is 5 x 10 ⁵ U / mol or more. The method according to claim 1, 2 or 3, wherein the polypeptide having catechol oxidase activity is tyrosinase. Filamentous tyrosinase selected from the group consisting of Aspergillus fungi, Neurospora fungi, Rhizomucor fungi, Tricoderma fungi, and Penicillium fungi The method according to claim 4, which is a fungal tyrosinase. The method according to claim 5, wherein the tyrosinase is a polypeptide encoded by the melB tyrosinase gene, melD tyrosinase gene or melO tyrosinase gene of Aspergillus oryzae. The method according to any one of claims 1 to 6, wherein the cell is a microbial cell selected from the group consisting of Escherichia coli, yeast and filamentous fungi. Furthermore, the composition adjustment process (A) which adjusts the composition by storing the melanin precursor obtained by an oxidation process in presence of a bivalent copper ion between an oxidation process and a collection | recovery process is included. 8. The method according to any one of 7. Furthermore, a composition adjustment step (B) for adjusting the composition by storing the melanin precursor obtained by the oxidation step in the presence of a buffer at −80 to 0 ° C. is included between the oxidation step and the recovery step. The method according to claim 1. Furthermore, the composition adjustment process (C) which adjusts the composition by preserving the melanin precursor obtained by an oxidation process in presence of a salt between an oxidation process and a collection | recovery process is any one of Claims 1-7 The method of crab. Furthermore, between the oxidation step and the recovery step, the melanin precursor obtained in the oxidation step is maintained at pH 3-5 in the absence of oxygen, pH 5-10 in the absence of oxygen, or pH 5-7 in the presence of oxygen. The method according to any one of claims 1 to 7 and 10, further comprising a composition adjustment step (D) for adjusting the composition by storage. Furthermore, the composition adjustment process (E) which adjusts the composition by storing the melanin precursor obtained by an oxidation process in presence of a water-soluble organic solvent between an oxidation process and a collection | recovery process is included. The method according to any one of -7, 10 and 11. When at least one substrate compound selected from the group consisting of tyrosine, DOPA, and analogs thereof is used, an enzyme having catechol oxidase activity has a catechol oxidase activity of 5 × when 1 mol of L-DOPA is used as a substrate. An oxidation step of oxidizing the substrate compound to convert it into a melanin precursor by acting so that it becomes 10 ⁵ U / mol or more;
The manufacturing method of a melanin precursor including the collection | recovery process which collect | recovers a melanin precursor from a reaction liquid. 14. The method according to claim 13, wherein the polypeptide having catechol oxidase activity is tyrosinase. Furthermore, the composition adjustment step (A) for adjusting the composition by storing the melanin precursor obtained by the oxidation step in the presence of divalent copper ions between the oxidation step and the recovery step is included. 14. The method according to 14. Furthermore, a composition adjustment step (B) for adjusting the composition by storing the melanin precursor obtained by the oxidation step in the presence of a buffer at −80 to 0 ° C. is included between the oxidation step and the recovery step. 16. A method according to claim 13, 14 or 15. Furthermore, the composition adjustment step (C) for adjusting the composition by storing the melanin precursor obtained by the oxidation step in the presence of a salt between the oxidation step and the recovery step is included in claim 13 or 14. the method of. Furthermore, between the oxidation step and the recovery step, the melanin precursor obtained in the oxidation step is maintained at pH 3-5 in the absence of oxygen, pH 5-10 in the absence of oxygen, or pH 5-7 in the presence of oxygen. The method according to claim 13, 14 or 17, further comprising a composition adjustment step (D) for adjusting the composition by storage. Furthermore, a composition adjustment step (E) for adjusting the composition of the melanin precursor obtained by the oxidation step by storing it in the presence of a water-soluble organic solvent is included between the oxidation step and the recovery step. , 14, 17 or 18. The melanin precursor obtained by the method in any one of Claims 1-19.

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