JP7504420B2 - Water-soluble carotenoid protein and its use - Google Patents

Description

IPOD FERM P-22374

The present invention relates to a novel water-soluble carotenoid protein that exhibits a red color. More specifically, the present invention relates to a carotenoid protein that exhibits water solubility and a red color when bound to astaxanthin.

Carotenoid is a general term for pigment compounds of carotenes and xanthophylls, and astaxanthin, a type of xanthophylls, is widely present in nature, such as microalgae, yeast, bacteria, and other microorganisms, fish such as salmon, salmon roe, and sea bream, crustaceans such as crabs, shrimp, and krill, and birds such as flamingos and chickens. Carotenoids have the property of imparting vivid colors to compositions, and are widely used as fat-soluble colorants in foods, health foods, medicines, and the like.
Carotenoids are also known to have strong antioxidant properties, and astaxanthin in particular has a higher singlet oxygen quenching activity than other antioxidants, and is attracting attention for its use in health foods, cosmetics, topical skin preparations, pharmaceuticals, etc.

Conventionally, astaxanthin is produced by enzymatically treating krill, drying and grinding the residue, and then purifying the extract with acetone. In recent years, a method for producing astaxanthin using microorganisms capable of stably producing large amounts of astaxanthin, such as microalgae, yeast, and bacteria, has been developed.
However, carotenoids including astaxanthin are fat-soluble and poorly soluble or insoluble in water, which limits their applications and methods of use in health foods, cosmetics, topical skin preparations, medicines, etc.

Here, the present inventors discovered AstaP-orange1, a water-soluble carotenoid-binding protein that selectively binds to astaxanthin, from microalgae belonging to the genus Coelastrella, a type of green algae (Patent Document 1). AstaP-orange1 binds to carotenoids such as astaxanthin to form a pigment protein, and exhibits the excellent property of enabling the use of water-insoluble astaxanthin as a water-soluble substance.
However, while AstaP-orange 1 is recognized to have great advantages in terms of the aqueous use of carotenoids, there is a problem in that its color-developing properties do not allow for sufficient scope of applications and practical application. One problem is that, in the field of food and beverages in recent years, there has been a demand for the development of astaxanthin-containing products that exhibit a bright reddish color that corresponds to a variety of color tones, but the protein tends to exhibit a yellowish orange color when in the form of a pigment protein bound to astaxanthin, and is not recognized as a color tone that is applicable to various products containing astaxanthin that exhibit a bright reddish color tone.

JP 2014-131992 A

The present invention was made in consideration of the above-mentioned problems in the prior art, and aims to provide a technology related to water-soluble carotenoid proteins that enables the use of astaxanthin in aqueous systems, and that enables astaxanthin to be used as a water-soluble pigment that exhibits a red hue.

In the above-mentioned state of the art, the present inventors conducted intensive research and discovered a new species of microalgae belonging to the genus Licorice in soil near a pond in Okinawa. When the microalgae was cultured under strong light and salt stress, it was found that a novel water-soluble carotenoid protein with a bright red hue was produced from the algal cells.
After further investigation, the inventors found that the carotenoid protein can be easily recovered from the water-soluble fraction of cultured algal cells, that it exhibits a bright red color as a pigment protein bound to astaxanthin, and that it has high water solubility and singlet oxygen quenching activity.
From this finding, it was discovered that by using this water-soluble carotenoid protein, astaxanthin can be used as a water-soluble pigment that exhibits a reddish hue, and that it can be used to color aqueous products (e.g., products with a high water content) in a reddish hue, in which astaxanthin could not be used with conventional technology.

The present inventors have completed the present invention based on the above findings. Specifically, the present invention relates to the following inventions.
[Item 1]
A carotenoid-binding protein having the characteristics described in (A), (B), and (C) below:
(A) (a-1) comprising the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 9, or (a-2) comprising an amino acid sequence in which one or more amino acid residues have been substituted, deleted, inserted, and/or added to the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 9,
(B) the carotenoid binding protein has the function of binding to astaxanthin to form a carotenoid protein;
(C) When the carotenoid binding protein forms a carotenoid protein, the carotenoid protein (c-1) exhibits the property of being soluble in water, (c-2) has singlet oxygen quenching activity when dissolved in water, and (c-3) exhibits a reddish color when dissolved in water.
[Item 2]
Item 2. The carotenoid-binding protein according to Item 1, wherein the amino acid sequence of (a-2) is an amino acid sequence that shows 85% or more sequence identity to the amino acid sequence shown in SEQ ID NO:8 or SEQ ID NO:9.
[Item 3]
Item 3. The carotenoid-binding protein according to item 1 or 2, which, when an aqueous solution is prepared by dissolving the carotenoid protein according to (C) in pure water, exhibits an absorption spectrum with a maximum absorption wavelength of 495 to 540 nm.
[Item 4]
A carotenoid-binding protein having the characteristic described in (A') below:
(A') The protein component is composed of the amino acid sequence shown in SEQ ID NO:8 or SEQ ID NO:9.
[Item 5]
Item 5. A water-soluble carotenoid protein comprising the carotenoid-binding protein according to any one of Items 1 to 4 bound to a carotenoid.
[Item 6]
Item 6. The water-soluble carotenoid protein according to Item 5, wherein the water-soluble carotenoid protein comprises, as a molecular population, a carotenoid protein bound to astaxanthin as a main molecule.
[Item 7]
A method for producing a water-soluble carotenoid protein, comprising the steps of:
Item 5. The method for producing a carotenoid, comprising the step of mixing the carotenoid-binding protein according to any one of Items 1 to 4 with a carotenoid.
[Item 8]
Item 7. An astaxanthin-containing composition comprising the water-soluble carotenoid protein according to item 5 or 6.
[Item 9]
Item 7. An astaxanthin-containing pigment composition, food or drink, cosmetic product, pharmaceutical product, quasi-drug product, sanitary daily necessities, feed, or reagent, comprising the water-soluble carotenoid protein according to Item 5 or 6.

The present invention provides a technology relating to a water-soluble carotenoid protein that enables the use of astaxanthin in aqueous systems, which enables astaxanthin to be used as a water-soluble pigment exhibiting a red hue. This makes it possible to use astaxanthin in a wide range of products, even in which it was difficult to use conventional astaxanthin due to its color-developing properties.

These are scanning electron microscope (SEM) photographs of the Oki-4N strain (Scenedesmus sp. Oki-4N) isolated in Experimental Example 1. The length of the bar shown under each photograph is 10 μm. This is a diagram showing a phylogenetic tree obtained by the neighbor-joining method based on the 18S rDNA base sequence in Experimental Example 1. In the diagram, "●" indicates the Oki-4N strain, and "○" indicates the Ki-4 strain. The length of the bar shown under the phylogenetic tree indicates the branch length corresponding to 0.2% base difference. In addition, the numerical value shown near each branch indicates the bootstrap value indicating the reliability of the branch. Fig. 3 is a diagram showing the results of observing the effect on the color tone of algal cells when the Oki-4N strain was cultured under salt stress in Experimental Example 2. Fig. 3A is a photographic image showing the time-dependent color change of algal cells observed under a microscope. Fig. 3B is a photographic image showing the water-soluble fraction obtained from the cell lysate. FIG. 1 shows the results of HPLC-photodiode array analysis of the water-soluble pigment fraction in Experimental Example 2. Fig. 5 shows experimental results for the carotenoid proteins purified in Experimental Example 3. Fig. 5A is a photographic image showing the color development state of fractions containing each carotenoid protein (chromoprotein). Fig. 5B is a photographic image showing the results of electrophoresis of each protein by SDS-PAGE. FIG. 13 shows the results of detecting the presence of sugar chains by PAS staining in a gel electrophoresed by SDS-PAGE in Experimental Example 3. FIG. 13 shows the results of detecting carotenoid compounds contained in each carotenoid protein (pigment protein) in Experimental Example 3. FIG. 13 shows the results of measuring the absorption spectrum of each carotenoid protein (chromoprotein) in pure water in Experimental Example 3. FIG. 13 shows the evaluation results of the singlet oxygen quenching activity of each carotenoid protein (chromoprotein) in Experimental Example 3. FIG. 1 shows the amino acid sequence and sequence characteristics of AstaP-orange2 in Experimental Example 4. Fig. 11 shows the amino acid sequence and sequence characteristics of AstaP-pink in Experimental Example 4. Fig. 11A shows information on AstaP-pink1. Fig. 11B shows information on AstaP-pink2. This is a diagram showing the results of constructing a gene phylogenetic tree by the neighbor-joining method for the amino acid sequences of genes belonging to the carotenoid-binding protein and fasciclin families in Experimental Example 4. The length of the bar shown under the phylogenetic tree indicates the branch length for 5% amino acid difference. In addition, the numbers shown near each branch indicate the bootstrap value indicating the reliability of the branch. FIG. 1 is a schematic diagram showing the primary structure of the amino acid sequence before processing of a carotenoid-binding protein in Experimental Example 4. FIG. 13 shows the results of detection of the mRNA expression pattern of the gene encoding the carotenoid-binding protein by Northern hybridization in Experimental Example 5. FIG. 13 shows the results of HPLC-photodiode array analysis of the water-soluble pigment fraction in Experimental Example 5. FIG. 13 shows the results of detecting intracellular localization of carotenoid proteins using a confocal fluorescence microscope in Experimental Example 5.

Hereinafter, an embodiment of the present invention will be described in detail.
The technical scope of the present invention is not limited to the embodiments including all of the configurations described below. Furthermore, the technical scope of the present invention does not exclude embodiments including configurations other than those described below.

In this specification, the term "carotenoid protein" refers to a pigment protein bound to a carotenoid such as astaxanthin, unless otherwise specified. That is, in this specification, the carotenoid protein refers to a molecular state in which a carotenoid-binding protein and a carotenoid are bound to each other.
In this specification, the term "carotenoid binding protein" refers to a protein having the function of binding to carotenoids such as astaxanthin, unless otherwise specified, and is used as a synonym for carotenoid-binding protein, protein having carotenoid-binding ability, etc. That is, in this specification, carotenoid-binding protein refers to a protein in a state not bound to a carotenoid. Furthermore, terms such as "AstaP-pink" and "AstaP-orange" indicating protein names are used as terms indicating proteins prior to binding to carotenoids such as astaxanthin, unless otherwise specified.
As used herein, the term "water-soluble protein" refers to a protein that is soluble in water.
As used herein, the term "sequence identity of **% or more" is used to indicate that, when sequences are compared, the percentage of elements (bases, amino acid residues) that make up these sequences that match is equal to or higher than a specified percentage.
In this specification, the term "protein component" is used as a term referring to a part of the protein body formed by polymerization of amino acids through peptide bonds. Furthermore, as the carotenoid binding protein or carotenoid protein according to the present invention, a molecular structure in which the amino acid residues of the protein component are molecularly modified with other molecules is acceptable. For example, a molecular structure in which the amino acid residues are molecularly modified with sugar chains or the like is also acceptable as the carotenoid binding protein or carotenoid protein according to the present invention. Furthermore, when these protein components form a three-dimensional structure, a molecular structure in which other molecules are coordinately bonded or bonded by hydrogen bonds or the like is also acceptable.
An embodiment of AstaP-pink, which is a carotenoid-binding protein according to the present invention, may include a protein structure that does not involve glycosylation of amino acid residues constituting the protein.

1. Water-soluble carotenoid protein The present invention relates to a novel water-soluble carotenoid protein that exhibits a red color. More specifically, the present invention relates to a carotenoid-binding protein that exhibits water solubility and a red color when bound to astaxanthin.

[Sequence features]
The protein body of the carotenoid protein according to the present invention is composed of a carotenoid binding protein.
The carotenoid-binding protein according to the present invention has the following sequence features:
(1) comprising the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 9, or (2) comprising an amino acid sequence in which one or more amino acid residues have been substituted, deleted, inserted, and/or added to the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 9. More preferably, the protein body is a protein whose constituent part (protein constituent part) is composed of the amino acid sequence.
The carotenoid-binding protein according to the present invention has carotenoid-binding properties, water solubility, etc., due to the sequence characteristics of the amino acid sequence of the protein. In addition, when bound to astaxanthin, functional characteristics related to singlet oxygen quenching activity and color development properties with a red hue are exhibited.

An example of the carotenoid-binding protein according to the present invention is a protein comprising the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 9. More preferably, the protein has a constituent part as the protein body, excluding the pigment compound, that is composed of the amino acid sequence.
Here, the amino acid sequences shown in SEQ ID NO:8 and SEQ ID NO:9 are the amino acid sequences constituting functional proteins isolated as mature proteins after processing of AstaP-pink1 and AstaP-pink2, respectively, in the Examples below. Here, AstaP-pink1 and AstaP-pink2 are homologous to each other, and show a high sequence identity of 89% (170/191 amino acid residues) when comparing the primary amino acid sequences before processing.

The carotenoid-binding protein according to the present invention is a protein belonging to the Fasciclin family, which has an H1 domain and an H2 domain as a sequence characteristic. The mature protein that functions as a carotenoid-binding protein has a structure in which the signal peptide has been removed. In addition, as a sequence characteristic, it has the property of having no glycosylation site. In addition, the isoelectric point pI value indicates a value that indicates an acidic protein. It preferably has a pI value of 3.5 to 5.5, more preferably a pI value of 3.6 to 4.8, and particularly preferably a pI value of 3.8 to 4.7.

The carotenoid-binding protein of the present invention can be a protein that comprises an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted, and/or added relative to the amino acid sequence of AstaP-pink1 or AstaP-pink2, as long as it maintains these functional characteristics.
Here, the substitution, deletion, insertion, and/or addition of amino acid residues is preferably a mutation that maintains or improves the function of the protein, but is acceptable as long as it does not substantially impair the function of the protein. Furthermore, in order to maintain the function of the protein, it is preferable that the amino acid residue after substitution has similar properties to the amino acid residue before substitution.
Furthermore, the mutation may include an amino acid sequence in which an amino acid residue has been added to the amino acid sequence of AstaP-pink1, 2. In other words, an amino acid sequence having additional amino acid residues on the N-terminal and/or C-terminal side of the main body portion that ensures the function of the protein is permissible as long as it does not substantially impair the function of the protein.

As for the carotenoid-binding protein of the present invention, there is no particular limitation on the degree of sequence mutation within the range of amino acid residue mutations as long as the function is ensured; however, it is desirable for the protein to have high sequence identity to the amino acid sequence of AstaP-pink1 or AstaP-pink2.
As a preferred embodiment of such a range showing high sequence identity in terms of ensuring function, a range of a protein containing an amino acid sequence showing 85% or more sequence identity to the amino acid sequence shown in SEQ ID NO:8 or SEQ ID NO:9 is suitable.
More preferably, the protein component has an amino acid sequence that shows 85% or more, 89% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 99.5% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 9. Here, the value of 89% sequence identity indicates the sequence identity between AstaP-pink1 and AstaP-pink2.
More preferably, the protein is composed of an amino acid sequence that shows high sequence identity.

[Functional features]
Due to the above sequence characteristics, the carotenoid-binding protein of the present invention has carotenoid-binding ability, and when bound to a carotenoid, becomes a protein with functions such as water solubility, singlet oxygen quenching activity, color development properties with a red hue, etc. This allows the protein to exhibit the functions of the water-soluble carotenoid protein (pigment protein) of the present invention.
The carotenoid-binding protein according to the present invention has the following functional characteristics:
The carotenoid-binding protein has a function of binding to astaxanthin to form a carotenoid protein,
When the carotenoid binding protein forms a carotenoid protein, the carotenoid protein (1) exhibits the property of being soluble in water, (2) has singlet oxygen quenching activity when dissolved in water, and (3) exhibits a reddish color when dissolved in water.

Carotenoid-binding property The carotenoid-binding protein according to the present invention is a protein having a binding ability to a carotenoid molecule. The carotenoid-binding protein binds to a carotenoid molecule to form a carotenoid protein, which is a pigment protein.
In this regard, the present invention includes an invention relating to a water-soluble carotenoid protein in which a carotenoid-binding protein is bound to a carotenoid.
Here, carotenoid refers to carotenes and xanthophylls, and is a fat-soluble compound that functions as a pigment compound or an antioxidant. Therefore, the carotenoid-binding protein according to the present invention can function as a pigment molecule by forming a molecular structure of a carotenoid protein bound to a carotenoid. Similarly, it can also exhibit singlet oxygen quenching activity.
Furthermore, the carotenoid protein may be in a state where other molecules besides the carotenoid molecule are further bound thereto, or in a state where other molecules are contained therein.

The carotenoid-binding protein according to the present invention has a selective binding ability to astaxanthin molecules with respect to its carotenoid-binding properties, i.e., the carotenoid-binding protein according to the present invention exhibits excellent binding ability to astaxanthin.
Here, as for carotenoid proteins in the form of pigment proteins, in a molecular population of homogeneous protein molecules produced in algal cells, astaxanthin-binding carotenoid proteins are the main molecules constituting the molecular population.
In this regard, in the present invention, the carotenoid protein (pigment protein) contains, as a molecular group, a carotenoid protein bound to astaxanthin (astaxanthin-binding type) as a main molecule.

Here, as for the carotenoid protein (pigment protein) according to the present invention, it is sufficient that astaxanthin-binding carotenoid protein constitutes the majority of the molecules contained in the molecular group (homologous protein). From the viewpoint of strongly exerting the function of astaxanthin, it is preferable that all of the molecules are of the astaxanthin-binding type. Taking into consideration the fact that the function of the carotenoid protein according to the present invention is guaranteed and the fact that it is produced from microalgae, if 70% or more, 75% or more, or 78% or more of the molecular group is of the astaxanthin-binding type, it can be suitably used as the water-soluble carotenoid-binding protein according to the present invention. From the viewpoint of more strongly exerting the function of astaxanthin, it is particularly preferable that 80% or more is of the astaxanthin-binding type.

As the carotenoid protein (chromoprotein) according to the present invention, a molecular population containing a binding type bound to other carotenoid molecules other than the astaxanthin binding type can be used. Here, the other carotenoid is not particularly limited as long as it does not substantially impair the overall function of the molecular population of the carotenoid protein according to the present invention, and examples thereof include lutein, adonixanthin, and canthaxanthin. Even in a molecular population of water-soluble carotenoid binding proteins containing binding types with these carotenoids as minor peaks, if functions such as singlet oxygen quenching activity and color development properties are sufficiently expressed, it can be used without problems as the carotenoid protein according to the present invention.
In one embodiment, the carotenoid other than astaxanthin as a bound type bound to a carotenoid molecule other than astaxanthin is lutein, adonixanthin, and/or canthaxanthin (it is permissible to include bound types of carotenoids other than these that are below the detection limit or have extremely small peaks in analysis, etc.), and as long as it accounts for 30% or less, particularly 20% or less of the molecular population of the carotenoid protein (homologous protein), it can be used without problem as the carotenoid protein of the present invention.

Water solubility: When the carotenoid-binding protein according to the present invention binds to a carotenoid to form a carotenoid protein (pigment protein), it exhibits excellent water solubility as a function of the protein. This property is exhibited by the sequence characteristics of the amino acid sequence constituting the protein. As a result, the main body of the protein becomes a water-soluble protein, and carotenoid molecules, which are originally difficult to use in aqueous systems, can be bound to the protein and dissolved in water for use.
More specifically, the carotenoid protein (chromoprotein) of the present invention has the following characteristics regarding its water solubility:
When the carotenoid protein is dissolved in pure water at 20° C. to prepare an aqueous solution, the carotenoid protein exhibits a water solubility of 5 mg/mL or more.

Here, the water solubility is more preferably 10 mg/mL or more, or 20 mg/mL or more. The pure water used to indicate the degree of water solubility as a standard refers to water that contains almost no metal ions or impurities, and an example of this is water with a resistivity of 18 MΩ cm or more.
The above-mentioned water solubility degree is not intended to mean that the water solubility of the carotenoid protein according to the present invention is exerted only in pure water. The water solubility of the carotenoid protein according to the present invention is a property that is exerted without any particular limitation in any aqueous solvent, such as not only ordinary water but also water containing salts or metals, aqueous buffer solutions, aqueous solutions containing organic compounds or various agents, etc., as long as the solvent is an aqueous solution.

The carotenoid-binding protein according to the present invention has singlet oxygen quenching activity as a function when bound to a carotenoid to form a carotenoid protein (pigment protein). This property is exhibited by the carotenoid (particularly astaxan) bound to the protein.
More specifically, the carotenoid protein according to the present invention has the following characteristics with respect to its singlet oxygen quenching activity:
A sample solution was prepared in 100 mM Tris-HCl buffer (pH 7.5) containing 0.3 μM of the carotenoid protein in terms of bound astaxanthin, 1 μM of rose bengal, and a singlet oxygen detection substance, and a control solution was prepared in the same manner except that it did not contain the carotenoid protein.When the sample solution and the control solution were irradiated with light at an intensity of 130 μmol/ ^m2 /s using a white fluorescent lamp at 25°C for 180 seconds, the sample solution showed a singlet oxygen quenching rate (%) of 30% or more compared to the control solution.

Here, the singlet oxygen quenching rate (%) can be calculated by the following formula (1) where the signal value from the detection substance for singlet oxygen in the sample solution is _S1 and the signal value from the control solution is _S0 .
Equation (1): Singlet oxygen quenching rate (%)=(1−S ₁ /S ₀ )×100
Here, as an example of a substance for detecting singlet oxygen, a substance that can detect the presence of singlet oxygen as a fluorescent signal can be used, such as SOSG (Singlet Oxygen Sensor Green, manufactured by Thermo Fisher Scientific), but is not particularly limited thereto.
The singlet oxygen quenching rate (%) is more preferably 34% or more, or 37% or more. In particular, the value is more preferably 40% or more. Also, the value shown in Table 3 or more is more preferably the same.
The above-mentioned singlet oxygen quenching rate (%) value indicates a singlet oxygen quenching activity equivalent to or greater than the singlet oxygen quenching rate (%) value measured in the same manner by the above-mentioned method using sodium azide, which is known to have singlet oxygen quenching activity (Examples of this specification).

Color-developing properties When the carotenoid-binding protein according to the present invention binds to a carotenoid to form a carotenoid protein (pigment protein), it has the function of exhibiting a color-developing property of exhibiting a reddish color when dissolved in water.
The color-developing property is a property that is exhibited by changing the original color-developing property of the carotenoid molecule by binding with the protein. Astaxanthin's color tone is originally a yellowish orange tone, but the color-developing property changes by binding with the protein, and the carotenoid protein of the present invention exhibits a color-developing property that shows a bright red tone.
More specifically, the carotenoid protein according to the present invention has the following characteristics regarding its color-developing properties:
When the carotenoid protein is dissolved in pure water to prepare an aqueous solution, the aqueous solution exhibits an absorption spectrum with a maximum absorption wavelength of 495 to 540 nm.
In this specification, the maximum absorption wavelength (λmax) refers to the light wavelength (nm) at which the absorbance of the dye composition in the visible light region is maximum.

Here, when the maximum absorption wavelength (λmax) of the absorption spectrum is 495 nm or more, it can be recognized as having a reddish color. Preferably, the value is 496 nm or more, 497 nm or more, 498 nm or more, 499 nm or more, or 500 nm or more, which is suitable for a reddish color tone as a color development characteristic. If the value is too low, the color tone will be yellowish, which is not suitable as the color tone required for the water-soluble carotenoid protein of the present invention. Most preferably, the maximum absorption wavelength (λmax) is 501 nm or more or 502 nm or more.
Furthermore, when the maximum absorption wavelength (λmax) is 540 nm or less, it can be recognized as having a reddish color. Preferably, the value is 535 nm or less, 530 nm or less, 525 nm or less, 520 nm or less, or 515 nm or less, which is suitable for a reddish color tone as a color development characteristic. If the value is too high, the color tone will be bluish, which is not suitable as the color tone required for the carotenoid protein of the present invention. Most preferably, the maximum absorption wavelength (λmax) is 510 nm or less or 505 nm or less.
Furthermore, the carotenoid protein according to the present invention is preferably one that exhibits the characteristic that no other peaks showing absorbance other than the peak at the maximum absorption wavelength are detectable in the visible light region in its spectral waveform.

The pure water used to demonstrate the color-developing properties may be the same as that described in the paragraph above regarding water solubility.
The red coloring characteristic of the carotenoid protein according to the present invention is not a color that can be exhibited only in pure water, but is a color that can be exhibited if the solvent is an aqueous solution of water. For example, not only normal water but also water containing salts or metals, aqueous buffer solutions, aqueous solutions containing organic compounds or various agents, and other solvents capable of dissolving the protein can be used to color the protein in a reddish color.

The coloring properties of the carotenoid protein of the present invention refer to a coloring property that exhibits a red tone, but variations in color tone due to saturation, brightness, etc. are permitted. For example, depending on the molecular structure and the conditions of the aqueous solution, color variations belonging to red tones such as pink, pearl red, wine red, rose red, and purple red are also possible.

2. Method for Producing Water-Soluble Carotenoid Protein An example of a method for producing the carotenoid protein of the present invention is to introduce a gene encoding the protein into Escherichia coli, yeast, or the like, and express the recombinant protein to produce the protein.
However, in consideration of production efficiency, etc., it is preferable to use microalgae as a method for efficiently producing the carotenoid protein of the present invention. In detail, the present invention includes an invention relating to a method for producing a water-soluble carotenoid protein, which comprises the following steps:
A process for culturing microalgae belonging to the genus Scenedesmus and having the characteristic of producing a water-soluble carotenoid protein having a reddish color as described in the above paragraph under salt stress in a medium containing sodium chloride.

[Microalgae producing water-soluble carotenoid proteins]
The production method of the present invention is characterized by utilizing a specific microalgae that has the characteristic of producing and accumulating an astaxanthin-binding water-soluble carotenoid protein that exhibits a reddish color in response to salt stress within its cells.
This characteristic is recognized as a phenomenon that occurs intracellularly in microalgae belonging to the genus Scenedesmus in response to environmental stress, and is recognized as a characteristic that is exhibited by the accumulation of mutations that enable a group of proteins belonging to the fasciclin family to function as a reddish-colored, astaxanthin-binding, water-soluble carotenoid protein.
That is, in the present invention, it is possible to use microalgae belonging to the genus Scenedesmus that have the characteristic of producing the above-mentioned carotenoid protein.
As a preferred embodiment of the microalgae, it is preferable to use microalgae that have the characteristic of producing a carotenoid-binding protein whose protein component is composed of the amino acid sequence shown in SEQ ID NO:8 or SEQ ID NO:9.

A suitable example of a microalgae having such characteristics is the microalgae belonging to the genus Scenedesmus, specifically the strain Oki-4N (Scenedesmus sp. Oki-4N). The Oki-4N strain is a new species of microalgae belonging to the genus Scenedesmus, family Scenedesmusaceae, class Chlorophyceae, isolated by the present inventors, and has been deposited at the National Institute of Technology and Evaluation, Japan Patent Organism Depositary under the accession number FERM P-22374.
That is, in the production method according to the present invention, Oki-4N strain (Scenedesmus sp. Oki-4N) can be suitably used as the microalgae having the above-mentioned characteristics.

Furthermore, in the production method of the present invention, it is possible to use microalgae derived from the Oki-4N strain that have the property of producing the above-mentioned carotenoid protein. Here, the microalgae derived from the Oki-4N strain include microalgae produced by subjecting the Oki-4N strain to mating, mutation, gene introduction, genome editing, or the like, and that retain the properties related to the above-mentioned carotenoid protein. Furthermore, the microalgae derived from the Oki-4N strain include progeny lines of the Oki-4N strain that are passages or self-fertilized progeny lines and that retain the property of producing the above-mentioned carotenoid protein.
These microalgae can be used in the production method of the present invention even if the Oki-4N strain has been imparted with or lacked additional traits, as long as the ability to produce the above-mentioned carotenoid protein is maintained.
Furthermore, in the production method according to the present invention, it is also possible to use microalgae that are further descendants of these and that have the property of producing the above-mentioned carotenoid protein.

The Oki-4N strain is not an artificially produced strain, but is a microalgae that originally existed in the natural environment, and therefore microalgae of the same species and with similar characteristics exist in the natural environment.
In this regard, the production method of the present invention makes it possible to use microalgae that belong to the same species as the Oki-4N strain and that have the ability to produce and accumulate the above-mentioned carotenoid protein within their cells.
An example of an indicator of the same species as the Oki-4N strain is a microalga that has a matching base sequence in the 18S rDNA (coding region of 18S ribosomal RNA) in its genome. Here, 18S rDNA is an indicator used in species-level biological classification, and a matching or substantially matching base sequence can be used as a basis for determining that the species is the same.
An example of the base sequence of the 18S rDNA of the Oki-4N strain is the base sequence set forth in SEQ ID NO: 14. This base sequence is contained in an 18S rDNA region having a base length of 1744 bp, and is a region having a base length sufficient to enable sequence comparison between species of the Botryllaceae family and sufficient to detect differences between species.
In other words, in the manufacturing method of the present invention, it is possible to use microalgae that have the base sequence shown in SEQ ID NO:14 in the 18S rDNA region of their genome with a sequence identity of 99.9% or more and that have the characteristics related to the above-mentioned carotenoid protein.
Here, the value of sequence identity of 99.9% is a difference (0.1% difference) that does not reach the base difference indicated by a branch length of 0.12% between the Oki-4N strain and the closely related species (Scenedesmus obtusus) that is the closest relative of the Oki-4N strain in the genus Scenedesmus in the phylogenetic tree shown in Figure 2 in the Examples below, and is a value that allows the Oki-4N strain to be distinguished from the closely related species in terms of base sequence.
Furthermore, with regard to the sequence identity, it is particularly preferred that the microalga has a sequence identity of 99.94% or more (0.06% difference: one base or less mutated, substantially identical) or 100% (perfect match) with the base sequence shown in SEQ ID NO:14 in the 18S rDNA region of its genome.

[Salt stress culture]
The microalgae that can be used in the production method of the present invention exhibit the property of producing and accumulating the carotenoid protein within the cells in response to salt stress from the growth environment, where the microalgae recognize culture in a medium containing a high concentration of sodium chloride as salt stress and exhibit the property of increasing the induction of expression of the carotenoid protein.
That is, the production method according to the present invention includes a step of culturing the microalgae under salt stress in a medium containing sodium chloride.
In this specification, the culture carried out in a medium containing a high concentration of sodium chloride in order to apply salt stress to the microalgae is referred to as salt stress culture or culture with salt stress applied.

The salt stress culture of the microalgae is preferably performed on cells in the proliferation stage by performing proliferation culture in a normal medium. The cells in the proliferation stage are in a state where the cells are actively dividing and the cell population is exponentially increasing, and therefore are suitable as cells to be used for the subsequent induction of protein production, etc.
That is, in the production method according to the present invention, it is preferable to carry out a salt stress culture on the microalgae after carrying out a growth culture (preculture or main culture, etc.) of the microalgae.
Here, the medium used for the growth culture may be a general medium that can be used for the growth culture of microalgae belonging to normal green algae, such as A-3 medium, TAP medium, etc., but is not limited thereto.
In addition, although the growth culture does not exclude culture on an agar medium, from the viewpoint of efficient protein production, it is preferable to carry out the growth culture by a culture method such as static culture, aeration culture, or agitation culture using a liquid medium.

The application of salt stress to perform salt stress culture can be started by adding sterilized sodium chloride to the growth culture medium in which the growth culture is being performed, or by separating the growth culture medium in which the growth culture is being performed from the cells by centrifugation, filtration, or the like, and replacing the medium with a medium containing sodium chloride at a high concentration.
The salt stress culture is desirably carried out until a sufficient amount of water-soluble carotenoid protein is produced and accumulated in the cells of the microalgae.
Specifically, the time for which the salt stress culture is carried out is preferably at least 12 hours, 1 day, or 2 days from the start of salt stress application, and more preferably 4 days or more from the start of salt stress application in terms of ensuring protein production.
The upper limit of the time for the salt stress culture can be set within a range in which the growth of the microalgae is not significantly inhibited, and is not particularly limited. One example is a mode in which the salt stress culture is performed for 30 days or less.

Specifically, the salt stress culture is carried out in a medium containing a high concentration of sodium chloride, which tends to inhibit the growth of normal green algae. The sodium chloride concentration is preferably 0.1 M or more, 0.2 M or more, or 0.25 M or more.
In the microalgae, in addition to the pigment protein having the above-mentioned red coloring property, another molecular species, an orange pigment protein, is also produced intracellularly as a water-soluble carotenoid protein. When the microalgae is cultured under salt stress with an increased salt concentration, the expression induction of the red water-soluble carotenoid protein increases, whereas the expression induction of another molecular, an orange water-soluble carotenoid protein, decreases.
In this regard, it is preferable to perform the salt stress culture of the microalgae at a sodium chloride concentration of 0.25 M or more, 0.3 M or more, 0.4 M or more, or 0.45 M or more. In particular, it is preferable to perform the salt stress culture at a sodium chloride concentration of 0.5 M or more, in which the production of a water-soluble carotenoid protein, which is a separate molecule with an orange hue, is significantly suppressed.
There is no particular upper limit to the sodium chloride concentration in the salt stress culture, as long as the growth of the microalgae is not significantly inhibited. One example is a mode in which salt stress culture is performed at a sodium chloride concentration of 2 M or less, 1.5 M or less, or 1 M or less.
The medium used for the salt stress culture can be the same as the normal growth medium used for growth culture, except that the sodium chloride concentration is adjusted to the above-mentioned concentration.
In addition, although the salt stress culture does not exclude culture on an agar medium, from the viewpoint of efficient protein production, it is preferable to carry out the culture by a culture method such as static culture, aeration culture, or agitation culture using a liquid medium.

The salt stress culture is preferably performed under culture conditions with strong light irradiation. In addition to the presence of high concentrations of sodium chloride, the microalgae exhibit the characteristic of producing and accumulating water-soluble carotenoid proteins within the cells as a stress response under culture conditions with strong light irradiation.
Specifically, in the salt stress culture, it is preferable to carry out the salt stress culture by irradiating with light having an intensity of 500 μmol/m ² /s or more.
The light intensity is more preferably 600 μmol/m ² /s or more or 800 μmol/m ² /s or more. There is no particular upper limit as long as the growth of the microalgae is not significantly inhibited, but an example is 2000 μmol/m ² /s or less or 1500 μmol/m ² /s or less.
Here, the light irradiation can be performed by white fluorescent light irradiation containing visible light and light of wavelengths around the visible light, or it is also possible to perform light irradiation using natural light, xenon, halogen, etc. as a light source. Also, if the wavelengths contained are appropriate, it is possible to use an LED as a light source.
As for the light irradiation conditions, continuous light irradiation is also possible, but light irradiation conditions in which a light-dark cycle is set are preferable in terms of the diurnal rhythm of living organisms. The light-dark cycle can be set under conditions that can be used in the cultivation of normal green algae. For example, conditions in which the light period is set to 6 to 16 hours, 8 to 14 hours, or 10 to 12 hours can be mentioned. The dark cycle can be set as the time obtained by subtracting the light period from 24 hours, if one cycle of the light-dark cycle is set to 24 hours.

The temperature conditions for the salt stress culture may be the general culture conditions used for culturing green algae. More specifically, the temperature may be 1 to 40°C, at which the above-mentioned microalgae can grow, and preferably 5 to 35°C. From the viewpoint of efficient protein production, the temperature may be preferably 15 to 30°C.
Other culture conditions for the salt stress culture can be adopted with reference to general culture conditions used in the culture of green algae, preferably with reference to culture conditions of other microalgae of the genus Lipodria.

After the salt stress culture, it is desirable to immediately carry out an operation to separate the carotenoid protein accumulated in the algal cells after the salt stress culture. It is also possible to temporarily return the cells to a normal medium after the salt stress culture and carry out a short period of culture, and then carry out an operation to separate the protein.

[Separation of red-hued water-soluble carotenoid proteins]
In the method for producing a water-soluble carotenoid protein according to the present invention, the cells after the above-mentioned salt stress culture are recovered, and a reddish water-soluble carotenoid protein is separated from the water-soluble fraction of the cells.
That is, the method for producing a water-soluble carotenoid protein according to the present invention includes a step of separating a fraction containing the water-soluble carotenoid protein from the cells of the microalgae that have been subjected to the salt stress culture.

(Pretreatment process)
In the production method according to the present invention, it is preferable to carry out, as a preliminary operation prior to the separation of the above-mentioned protein, an operation of recovering the algal cells after the above-mentioned salt stress culture and obtaining a water-soluble fraction contained within the cells of the algal cells.
The means for recovering the algal cells can be any means capable of achieving a high cell density, without particular limitation. For example, the recovery can be performed by a conventional method used for recovering cells, such as centrifugation or filtration.

The water-soluble fraction contained within the collected algal cells is separated and obtained. The separation of the water-soluble fraction from the algal cells can be performed by a conventional method as long as it is possible to recover the aqueous solution within the cells, which is the content, by destroying and/or decomposing the cell walls. Examples include a method using a French press, a homogenizer, ultrasonic irradiation, a cell wall decomposing enzyme, etc., but in an embodiment assuming mass production, it is preferable to use a method of cell disruption (e.g., a method using a pressure cell disrupter, etc.). When performing cell disruption here, it is preferable to add a protease inhibitor or the like before the cell disruption operation in order to prevent protein decomposition.
After the cell wall destruction or decomposition, it is desirable to carry out an operation such as centrifugation or filtration to remove cell debris and impurities.

The water-soluble fraction from the algal cells obtained by this process is an aqueous solution (water-soluble pigment fraction) that contains a large amount of water-soluble pigment proteins. However, at this separation stage, in addition to the water-soluble carotenoid proteins that exhibit the desired red color, the solution also contains water-soluble carotenoid proteins, which are separate molecules that exhibit an orange color, and other pigment compounds, and the overall color of the aqueous solution is a slightly darker orange or orange color.

(Refining process)
The production method according to the present invention includes a step of separating a fraction containing a water-soluble carotenoid protein exhibiting a desired red color from the above water-soluble fraction.
The separation step can be carried out using any conventional technique without particular limitation, as long as it is possible to obtain a fraction containing a water-soluble carotenoid protein exhibiting the desired red color by separating it from other pigment proteins or pigment compounds.
The separation step does not exclude electrophoretic separation techniques such as SDS-PAGE, but in consideration of the recovery efficiency and degree of purification of the protein, a suitable example is a technique in which a protein purification treatment is performed using a gel filtration column and/or an ion exchange resin column, etc., to obtain a desired protein fraction.

More specifically, the separation step by column purification is performed by purifying the protein using a gel filtration column, which allows the separation of impurities such as other chromoproteins and pigment compounds having different molecular weights, and allows the separation of a purified fraction containing a water-soluble carotenoid protein exhibiting a desired red hue. The purified fraction obtained by the purification process removes other carotenoid proteins exhibiting an orange color having different molecular weights (e.g., chromoproteins in which AstaP-orange2 and a carotenoid are bound), other chromoproteins, low molecular weight compounds, etc., and therefore allows the fraction to be separated as a fraction containing a carotenoid protein exhibiting a red hue. The purified fraction exhibits a red hue as the overall color tone of the aqueous solution. The purified fraction may contain two types of water-soluble carotenoid proteins exhibiting a red hue that have similar molecular weights (e.g., chromoproteins in which AstaP-pink1 or AstaP-pink2 and a carotenoid are bound).
In addition, the separation step by column purification can be performed in an embodiment in which protein purification is performed using an ion exchange resin column. In this embodiment, it is possible to separate the water-soluble carotenoid protein at the point of its isoelectric point to obtain a purified fraction. Specifically, separation by gradient elution with sodium chloride or the like is possible using an anionic resin (DEAE resin, etc.). In the purified fraction, it is possible to separate and obtain two water-soluble carotenoid proteins exhibiting red tones with different isoelectric points (for example, a pigment protein in which AstaP-pink1 or AstaP-pink2 is bound to a carotenoid). In this purification, other carotenoid proteins exhibiting orange with different isoelectric points (for example, a pigment protein in which AstaP-orange2 is bound to a carotenoid), other pigment proteins, low molecular weight compounds, etc. are also removed, so that the purified fraction exhibits a red tone as the color tone of the entire aqueous solution.
In addition, as the separation step by column purification, it is more preferable to perform both purification on an ion exchange resin column and purification on a gel filtration column to obtain a purified protein fraction with an increased degree of purification.

[Other processes, etc.]
The method for producing a water-soluble carotenoid protein according to the present invention can be embodied in such a manner that, in addition to the above steps, a step of carrying out a treatment or the like as desired is included.
For example, it is possible to obtain a water-soluble carotenoid protein in a desired state and/or form by carrying out a desalting treatment, a dialysis treatment, a purification treatment, a dilution treatment, a concentration treatment, a drying treatment, etc. These steps can be carried out in combination with any desired treatment, and any desired treatment can be carried out multiple times.
The water-soluble carotenoid protein according to the present invention is in a liquid form in which the purified protein is dissolved in a liquid after the above-mentioned purification and separation step, but can be made into various forms such as liquid, paste, powder, solid, etc. by carrying out various treatments. Also, it can be made into a form of a composition containing the water-soluble carotenoid protein.

[Preparation of desired carotenoid proteins]
Furthermore, as the water-soluble carotenoid protein of the present invention, it is possible to prepare a carotenoid-binding protein (a protein not bound to a carotenoid) and a carotenoid, and mix these to produce a carotenoid protein in which the two are bound.
That is, the present invention includes an invention relating to a method for producing a water-soluble carotenoid protein, which comprises a step of mixing a carotenoid binding protein with a carotenoid.
Here, the carotenoid-binding protein not bound to the carotenoid can be obtained by separating the carotenoid from the carotenoid protein derived from the algae. The separation method can be, for example, the Bligh & Dyer method, but is not particularly limited thereto. In addition, it is also possible to introduce a gene encoding the carotenoid-binding protein into Escherichia coli, yeast, or the like, and obtain it as a recombinant protein.
The mixing step can be carried out in an aqueous solution, i.e., in this step, the carotenoid binding protein and the carotenoid are mixed in an aqueous solution, thereby making it possible to form a carotenoid protein in which the two are bound to each other.

This production method makes it possible to utilize the selective astaxanthin-binding ability of the carotenoid binding protein of the present invention, and therefore, even if the carotenoid used in the mixing process contains multiple carotenoid molecular species, it is possible to produce a carotenoid protein whose molecular population contains a high concentration of astaxanthin-binding carotenoid protein.
Furthermore, in this production method, when only a desired carotenoid is used as the carotenoid used in the mixing step, it is possible to prepare a carotenoid protein that is a molecular population composed of a carotenoid protein bound to the desired carotenoid.
That is, this method makes it possible to produce a carotenoid protein whose molecular population is composed of a desired carotenoid protein or contains the desired carotenoid protein at a very high purity. As the carotenoid molecule used in this embodiment, astaxanthin is preferably used, but it is also possible to select and use a desired carotenoid other than astaxanthin (for example, the carotenoids described above, such as lutein).

3. Astaxanthin-Containing Composition The water-soluble carotenoid protein according to the present invention is a protein in which the water-soluble carotenoid protein that is of astaxanthin-binding type is the main molecule in the molecular population. Therefore, a composition containing the water-soluble carotenoid protein can be used as an astaxanthin-containing composition.

The present invention includes an invention related to a method for producing an astaxanthin-containing composition. The method is characterized by blending or containing the above-mentioned water-soluble carotenoid protein. In the method, the desired processing can be performed in the production process depending on the intended use. There is no particular limitation on the various processing treatments as long as they do not substantially impair the effects of the present invention, but depending on the intended use, a composition with the desired quality and/or form can be obtained by performing solid-liquid separation, purification, concentration, dilution, pH adjustment, drying, sterilization, etc. Furthermore, in the production of products such as color preparations that require deodorization due to the components contained therein, further purification treatments, etc. are also possible.
In the manufacturing process, desired treatments may be combined, and desired treatments may be performed multiple times.

Furthermore, the astaxan-containing composition according to the present invention may be formulated to contain other compounds or functional ingredients, etc., as long as the color-developing properties and protein functions are not substantially impaired. For example, excipients, antioxidants, pH adjusters, thickening polysaccharides, flavoring compounds, and other food ingredients may be contained or formulated, but are not limited to these.
Furthermore, the astaxan-containing composition according to the present invention can be made into compositions specialized for various applications as shown below.

[Dye composition]
The water-soluble carotenoid protein according to the present invention can be used as a pigment composition containing astaxanthin.
That is, the present invention includes an invention relating to a pigment composition or pigment preparation containing astaxanthin, which contains the above-mentioned water-soluble carotenoid protein. Also included is an invention relating to a method for producing a pigment composition or pigment preparation containing astaxanthin.
The dye composition according to the present invention may be a liquid composition or a solid composition containing the water-soluble carotenoid protein. In addition, the dye composition may be in a state of being processed into a dye preparation or the like. These may be used to color foods, beverages, and various other products.
In this specification, the term "dye composition" can be read as "dye preparation" depending on the content.

The form of the dye composition according to the present invention is not particularly limited, but examples thereof include liquid, paste, gel, semi-solid, solid, powder, etc. Also included are processed solid forms such as granules and tablets.
The dye composition according to the present invention can be used as an aqueous solution or a water-soluble dye preparation dissolved in water.

The content of the water-soluble carotenoid protein in the pigment composition according to the present invention is not particularly limited, and may be any content that takes into consideration the final concentration in the form of use.For example, the content of the water ^- soluble _carotenoid protein in the composition may be, in terms of color value, 0.0001 or more, 0.001 or more, or 0.005 or more, and more preferably 0.01 or more, in terms of color value conversion.In this case, the upper limit of the color value is not particularly limited, but may be, for example, 200 or less or 50 or less.
In another preferred embodiment of the present invention, when the dye composition is a dye preparation, the dye composition may have a color value of 10 or more, 50 or more, or 100 or more. There is no particular upper limit to the color value of the dye composition according to the present invention, but the upper limit may be, for example, 800 or less, or 500 or less.

[Various products]
From the viewpoint of its water solubility and color development properties, the water-soluble carotenoid protein of the present invention can be used in a wide range of applications and products in which the use of astaxanthin has been difficult using conventional techniques.
That is, the present invention includes inventions relating to various products containing astaxanthin, which contain the above-mentioned water-soluble carotenoid protein. The present invention also includes inventions relating to methods for producing various products containing astaxanthin.

The product according to the present invention is not particularly limited in terms of its product form or specific embodiment, but in particular, the present invention makes it possible to suitably provide various products, such as food and beverages, cosmetics, pharmaceuticals, quasi-drugs, daily sanitary products, and feed, which contain astaxanthin that can be used in aqueous systems and has a reddish color.
Examples of products that can be colored with astaxanthin according to the present invention are shown below, but the products that can be colored according to the present invention are not limited to these.
Examples of "food and beverages" include beverages, frozen desserts, desserts, sugar confectioneries (e.g., candy, gummy candies, marshmallows), gum, chocolate, confectioneries (e.g., cookies, etc.), bread, processed agricultural products (e.g., pickles, etc.), processed meat products, processed seafood products, dairy products, noodles, seasonings, jellies, syrups, jams, sauces, and alcoholic beverages.
Examples of "cosmetics" include perfumes, cosmetics, skin care products, toiletry products, and the like, such as skin lotions, lipsticks, sunscreen cosmetics, and makeup cosmetics.
Examples of "pharmaceuticals" include various tablets, capsules, drinks, lozenges, mouthwashes, and the like.
Examples of "quasi-drugs" include nutritional supplements, various supplements, toothpaste, mouth fresheners, odor control agents, hair care products, hair restorers, skin moisturizers, and the like.
Examples of "hygienic daily necessities" include soaps, detergents, shampoos, rinses, hair treatments, toothpastes, bath additives, etc.
Examples of "feed" include various pet foods such as cat food and dog food; feed for ornamental fish and farmed fish; and the like.

[reagent]
Since the water-soluble carotenoid protein according to the present invention is a protein molecule containing astaxanthin as the main carotenoid, the purified protein can be used as a product form of a reagent containing astaxanthin.
That is, the present invention includes an invention relating to a reagent containing astaxanthin, and also includes an invention relating to a method for producing a reagent containing astaxanthin.

The form of the reagent according to the present invention is not particularly limited to these, but examples thereof include powder, solid, liquid, paste, etc. Also included are processed solid forms such as granules and tablets.
The reagent according to the present invention can be used as an aqueous solution or as an aqueous reagent dissolved in water.

The present invention will be described below with reference to examples, but the scope of the present invention is not limited to these examples.
In the following Examples, the symbol "ND" in the tables showing the results of signal detection etc. indicates that the detected signal was below the detection limit.
The A-3 medium used in the following examples is a medium having _a composition containing 125 mg of _KNO3 , ₇₅ mg of MgSO4.7H2O, ₇₅ mg of _K2HPO4 , ₁₇₅ mg _of KH2PO4, 25 _{mg of} NaCl, ₁₀ mg of _CaCl2.H2O , ₁ mg of _FeSO4.7H2O , ₃ mg of H3BO3, 2.5 _mg of MnSO4.7H2O, 2 mg of _ZnSO4.7H2O , 0.1 mg of _CuSO4.5H2O , and _0.1 mg of _NaMoO4 in ₁ L of pure water. In the case of an agar plate medium, the composition further contains 15 g of agar.

[Experimental Example 1] "Isolation and identification of microalgae that produce water-soluble carotenoid proteins"
Microalgae producing water-soluble carotenoid proteins were isolated from samples collected in the field, and their biological properties were evaluated.

(1) Isolation of microalgae Soil from the surface of a reservoir in Okinawa was collected, spread on the surface of an A-3 agar plate medium, and cultured in a static environment under natural lighting conditions indoors. Green colonies that appeared to be microalgae growing on the agar plate medium were then collected, and the process of re-culture and colony collection was repeated until the microalgae forming the colonies were single, yielding a pure culture strain of the microalgae.
From the many isolated microalgae strains, an algae strain that was capable of growing even under culture conditions of strong light and salt stress and turned orange was selected. This selected strain was named Oki-4N.

(2) Biological Characteristics of Microalgae The biological characteristics of the isolated Oki-4N strain were evaluated.
When the microalgae were observed under a scanning electron microscope (SEM), they were found to be unicellular algae with an elliptical shape (Figure 1). The selected strain had green cells under normal culture conditions, but showed a characteristic of turning the inside of the cells orange when cultured under stress conditions of strong light and high salt concentration (culture conditions described in Experimental Example 2).

As shown by the analytical results described below, the orange-colored cells of the Oki-4N strain were microalgae cells that expressed the carotenoid-binding proteins AstaP-orange2, AstaP-pink1, and AstaP-pink2. Of these, AstaP-pink1 and AstaP-pink2 were pigment proteins that bound to astaxanthin and exhibited a red hue. Furthermore, these pigment proteins were water-soluble proteins present in the Oki-4N strain cells.

DNA was extracted from the Oki-4N strain, and PCR amplification was performed using a universal primer set (sequence numbers 12 and 13) capable of amplifying 18S rDNA (the coding region of 18S ribosomal RNA). The base sequence of the 18S rDNA of the algae strain was then determined using a capillary DNA sequencer (3730xl DNA analyser, Applied Biosystems).
As a result, it was shown that the 18S rDNA of the Oki-4N strain is a DNA region having the 1,744 bp base sequence shown in SEQ ID NO:14.
A homology search was performed on the 18S rDNA of the Oki-4N strain in the DDBJ (National Institute of Genetics DNA Data Bank), and no sequence identical to the sequence was registered, showing sequence similarity with algae of the family Botryococcus of the Chlorophyceae class.

Next, multiple alignment was performed using the 18S rDNA sequence data of the Oki-4N strain and the 18S rDNA sequence data of other microalgae, and phylogenetic analysis was performed by the neighbor-joining method (MEGA version 7.0.2) in the aligned regions. A bootstrap test to show reliability was performed 1000 times. The obtained phylogenetic tree is shown in Figure 2. Oki-4N is indicated by a "●" in the phylogenetic tree.
As a result, it was shown that the Oki-4N strain is included in a group of microalgae belonging to the genus Scenedesmus in the strict sense of the family Scenedesmus with a bootstrap value of 98% (Figure 2). In addition, the branch length of the phylogenetic tree showed that there are sequence differences at the species level from known members of the genus Scenedesmus.
Furthermore, the Ki-4 strain (Patent Document 1, 18S rDNA indicated by "○" in the phylogenetic tree in FIG. 2, SEQ ID NO: 15), which is a producer of AstaP-orange1 previously reported by the present inventors, has been recently reclassified as a microalga classified in the genus Coelastrella, whereas the Oki-4 strain has been shown to be classified in a different genus from the Ki-4 strain.
From the above results, it was shown that the Oki-4N strain is a "new species" of microalgae belonging to the Chlorophyceae, Scenedesmaceae, Scenedesmus genus.

(3) References to the deposited biological material The above-mentioned isolated Scenedesmus sp. Oki-4N strain was applied for deposit at the National Institute of Technology and Evaluation, Patent Organism Depositary Center on May 24, 2019, and was assigned the deposit number "FERM P-22374". A certificate of deposit and a certificate of viability were issued for the biological material on August 5, 2019.

[Depository institution]
Name: National Institute of Technology and Evaluation, Patent Biological Deposit Center Address: Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818 [Date of deposit]
Date of deposit (acceptance): May 24, 2019 Date of receipt notification: August 5, 2019 Date of live certificate notification: August 5, 2019 [Depositor]
Name or name of the person: Shinji Kawasaki Address: 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502 Department of Molecular Microbiology, Tokyo University of Agriculture [References to the deposit]
The depositor has granted applicant permission to refer to the deposited organisms in this application, and has given applicant his consent that the deposited organisms will be made available to the public.
[Accession number]
FERM P-22374: Scenedesmus sp. Oki-4N
[Characteristics of the deposited organism]
The characteristics of the deposited organism related to FERM P-22374 are as described in the description of the present specification and in the Examples.
In addition, the deposited organism related to FERM P-22374 has the following characteristics:
- They are green and consist of single cells (unicellular algae) that do not form colonies.
- They are elliptical in shape, about 5 to 20 μm.
-Chloroplasts contain pyrenoids.
- It is light-requiring.
- Conditions suitable for vegetative growth: pH 6.0-6.5, 25-30°C, TAP medium, A-3 medium, etc. - Under salt stress culture conditions, it exhibits the property of inducing the expression of carotenoid-binding proteins AstaP-orange2, AstaP-pink1, and AstaP-pink2. It also exhibits the property of accumulating water-soluble carotenoid proteins in which these carotenoid-binding proteins and carotenoids are bound within the cells.

[Experimental Example 2] "Separation of water-soluble pigment fraction"
A water-soluble pigment fraction was separated from the isolated Oki-4N strain and its characteristics were investigated.

(1) Salt stress culture For the Oki-4N strain isolated above, A-3 liquid medium (5 L) was placed in an Erlenmeyer flask fitted with a cotton-plugged glass tube, and agitation culture was performed using a stirrer bar at 28°C under a light intensity of 800 μmol/m ² /s and a photoperiod of 16 hours light and 8 hours dark, to obtain a culture solution with a cell concentration showing an _O.D.750 value of 1.0.
NaCl was added to the culture solution to a final concentration of 0.5 M (A-3 medium containing 0.5 M NaCl), and agitation culture was performed under the same conditions. The culture in the presence of high concentrations of NaCl was performed as a culture for applying salt stress.
Here, the cells in the culture medium before salt stress (Test 2-1) were green, whereas the cells after salt stress (Test 2-2, Test 2-3) showed a tendency to turn orange in the Oki-4N strain cells. Furthermore, the color tone became darker after 4 days of salt stress (Figure 3A).

(2) Separation of Water-Soluble Pigment Fraction After the salt stress culture in the salt stress culture, the algae cells were collected by centrifugation. The collected algae cells (100 mg wet cells/ml) were suspended in 50 mM Tris-HCl buffer (pH 7.5) containing a protease inhibitor, and then the cells were disrupted at 140 MPa using a pressure cell disrupter (FRENCH PRESSURE 30,000 psi, manufactured by AMINCO), and the cell residues were removed by centrifugation. Then, the cells were centrifuged (100,000×g) using an ultracentrifuge to collect the supernatant, and the water-soluble fraction from the microgreen algae was obtained.
The absorbance of these water-soluble fractions at a wavelength of 480 nm was measured, and the results are shown in the table below. As a result, it was shown that the water-soluble fractions from the cells cultured under salt stress (Test 2-2, Test 2-3) contained a large amount of a pigment absorbing at a wavelength of 480 nm, and the water-soluble fraction from the cells cultured under salt stress for 4 days (Test 2-3) contained a larger amount of a pigment than the water-soluble fraction from the cells cultured under salt stress for 2 days (Test 2-2).
Furthermore, when the test tubes containing the water-soluble fractions were visually observed, the water-soluble fraction from the cells before salt stress (Test 2-1) was transparent, whereas the water-soluble fractions after salt stress (Test 2-2, Test 2-3) were orange in color (Figure 3B).

(3) HPLC-photodiode array analysis of water-soluble pigment fraction The water-soluble pigment fraction obtained above was analyzed using an HPLC-photodiode array detector (Hitachi). In the photodiode array detection, the relative signal intensity of each peak having absorbance in the visible light wavelength range (around 400 to 800 nm) was detected for the fractions separated by HPLC according to molecular weight. The results are shown in the following table and FIG. 4.
As a result, it was shown that two peaks with different molecular weights (Peak 2 and Peak 3) were detected in the water-soluble pigment fraction from cells cultured under salt stress (Test 2-2 and Test 2-3), which were not detected in the water-soluble pigment fraction from cells before salt stress (Test 2-1). Peaks 2 and 3 were presumed to be water-soluble pigment proteins having a maximum absorption wavelength in the vicinity of 450 to 550 nm, based on the distribution of their detection signal intensities.
Furthermore, it was shown that these two peaks (peak 2 and peak 3) were detected with increased signal intensity in the water-soluble pigment fraction from cells cultured under salt stress for 4 days (Test 2-3) compared to the water-soluble pigment fraction from cells cultured under salt stress for 2 days (Test 2-2).

[Experimental Example 3] "Purification of carotenoid protein"
The water-soluble chromoproteins contained in the water-soluble fraction were isolated and purified, and the properties of each purified protein were evaluated.

(1) Protein isolation and purification The water-soluble fraction obtained by cell disruption in Experimental Example 2(2) was applied to a DEAE Sepharose column (DEAE Sepharose Fast Flow, GE Healthcare Japan) equilibrated with 50 mM Na-Pi buffer (pH 5.0), and gradient elution was performed with a linear concentration gradient (0 to 300 mM NaCl) using 50 mM Na-Pi buffer (pH 5.0) containing NaCl to separate protein fractions based on differences in charge. Proteins were detected by monitoring at a wavelength of 280 nm.

As a result, three fractions with different binding strength to the anionic resin carrier were separated from the DEAE column resin adsorption fraction. Each of these fractions was purified using a gel filtration column (HR100, GE Healthcare Japan, Inc.) to obtain purified protein fractions. Photographs of the colors of each purified protein fraction are shown in Figure 5A.
Two of these fractions contained a chromoprotein with a pinkish red hue (purified fractions P1 and P2), while the remaining fraction contained a chromoprotein with a yellowish orange hue (purified fraction O).

The purification state and molecular weight of each of the proteins purified above were confirmed by CBB staining using SDS-PAGE. The results are shown in Figure 5B.
As a result, it was confirmed that these proteins were purified as single proteins. The water-soluble proteins contained in the purified fractions O, P1, and P2 were named AstaP-orange2, AstaP-pink1, and AstaP-pink2, respectively.
The estimated molecular weights of these proteins by SDS-PAGE were 75-100 kDa, 15-20 kDa, and 15-20 kDa, respectively.
From the results of the molecular weight estimation, AstaP-orange2 was recognized as the protein contained in peak 2 of Experimental Example 2(2). In addition, AstaP-pink1 and AstaP-pink2 were recognized as the proteins contained in peak 3 of Experimental Example 2(2).

(2) Analysis of Glycosylation Each of the proteins purified above was subjected to SDS-PAGE in the same manner as described above, and the glycans of the resulting gel were detected by the PAS staining method. In lane C, a known glycoprotein was used as a positive control sample. The results are shown in Figure 6.
As a result, color development by the Schiff's reagent was confirmed in the band showing AstaP-orange 2 (lane O), indicating that AstaP-orange 2 is a glycoprotein having a sugar chain.
On the other hand, no color development by Schiff's reagent was observed in either band representing AstaP-pink1 or AstaP-pink2 (lanes P1 and P2), indicating that AstaP-pink1 and AstaP-pink2 are non-glycosylated proteins.

(3) Identification of bound dyes For each of the chromoproteins purified above, the elution time and spectrum of each chromoprotein was compared with that of a standard compound using an HPLC-photodiode array detector (Hitachi). Next, the chromoproteins were subjected to analysis using a high-resolution LC-MS device (Shimadzu Corporation) to identify the chromoproteins bound to the protein moiety that constitutes the chromoprotein. The results are shown in FIG. 7.
As a result, a clear and large peak Ax corresponding to a molecular weight of 596 was detected in all of the proteins AstaP-orange2, AstaP-pink1, and AstaP-pink2. As a result of comparison with peaks of standard compounds, this peak was identified as astaxanthin, a type of carotenoid pigment.
In addition, trace amounts of carotenoid pigments identified as adonixanthin with a molecular weight of 582, shown as peak Ad, lutein with a molecular weight of 568, shown as peak Lt, and canthaxanthin with a molecular weight of 564, shown as peak Ca, were detected as pigment compounds binding to these proteins.
These results demonstrated that AstaP-orange2, AstaP-pink1, and AstaP-pink2 are carotenoid-binding proteins capable of binding to carotenoids. It was also revealed that most of these carotenoid-binding proteins exist as pigment proteins bound to astaxanthin. The peak Ax representing astaxanthin was 78% for AstaP-orange2, 80% for AstaP-pink1, and 82% for AstaP-pink2 relative to the sum of the peak heights of the four main peaks.

(4) Spectral waveform showing color development characteristics Each of the pigment proteins (carotenoid proteins) purified above was adsorbed onto a PD-10 desalting column (GE Healthcare), eluted with ultrapure water (MilliQ MERCK, resistivity 18 MΩ cm), and the absorption spectral waveform was measured at measurement wavelengths of 250 to 600 nm using a spectrophotometer. The obtained waveform results are shown in FIG.
As a result, the carotenoid protein of AstaP-orange2 exhibited a spectral waveform showing a maximum absorption peak at a wavelength of 488 nm. Meanwhile, the carotenoid proteins of AstaP-pink1 and AstaP-pink2 exhibited spectral waveforms showing maximum absorption peaks at wavelengths of 502 nm and 501 nm, respectively. Furthermore, no other peaks showing absorption other than the maximum absorption peak were detected in the visible light range of 350 to 600 nm in these spectral waveforms.
The spectral waveform results were consistent with the color development characteristics in which the fraction containing AstaP-orange2 exhibited a yellowish orange color, and the fraction containing AstaP-pink1 or AstaP-pink2 exhibited a bright pink red color.

(5) Water solubility test The solvent in each solution containing each purified carotenoid protein was replaced with ultrapure water using a PD-10 desalting column (GE Healthcare) and dialysis. The protein concentration was adjusted to about 0.2 mg/mL, and 1 mL of the solution was vacuum dried to remove water and obtain a dry powder.
To these, 10 μL of ultrapure water (MilliQ MERCK, resistivity 18 MΩ cm) was added at 20° C. As a result, it was confirmed that the dry powder quickly dissolved, no insoluble matter was observed in the solution, and each solution became an aqueous solution of each color tone observed in (1) above. The results showed that the water solubility of these carotenoid proteins was 20 mg/mL or more.

(6) Evaluation of singlet oxygen quenching activity 500 μL of a sample solution was prepared in 100 mM Tris-HCl buffer (pH 7.5) containing 0.3 μM of each of the purified carotenoid proteins in terms of bound astaxanthin, 5 μM of SOSG (Singlet Oxygen Sensor Green, Thermo Fisher Scientific), a fluorescent substance capable of detecting singlet oxygen, and 1 μM of rose bengal, a singlet oxygen source.
As a comparative sample solution, a Tris-HCl buffer solution (pH 7.5) was prepared in the same manner as above, except that sodium azide (NaN ₃ ), which has singlet oxygen quenching activity, was contained at 500 μM instead of the carotenoid protein.
As a control solution not containing a substance having singlet oxygen quenching activity, a Tris-HCl buffer solution (pH 7.5) was prepared in the same manner as above, except that it did not contain the carotenoid protein and sodium azide.
Each sample solution and control solution was irradiated with light at an intensity of 130 μmol/ ^m2 /s for 180 seconds at 25°C using a white fluorescent lamp (EFD21ED, 400 nm-650 nm, Toshiba), and then the fluorescence intensity was measured using a spectrofluorometer (RF6000 Spectrofluorometer, Shimazu) with excitation light at 488 nm and fluorescence at 525 nm.
The signal intensity of each sample solution was taken as S ₁ , and the signal intensity of the control solution was taken as S ₀ , and the singlet oxygen quenching rate was calculated according to the above formula (1). The results are shown in the following table and FIG.

As a result, it was shown that an aqueous solution containing each of the carotenoid proteins purified above at a concentration of 0.3 μM exhibited significantly higher singlet oxygen quenching activity than an aqueous solution containing sodium azide at a concentration of 500 μM.
In particular, AstaP-pink1 and 2 exhibited higher activity than other carotenoid proteins, exhibiting singlet oxygen quenching activity 1.8 times or more that of an aqueous solution containing sodium azide at a concentration of 500 μM.

[Experimental Example 4] "Sequence characteristics of carotenoid-binding proteins"
The amino acid sequence of the carotenoid binding protein, which is the main protein of the purified carotenoid protein described above, was determined, and knowledge was obtained from the sequence information.

(1) Creation of a cDNA Library The isolated Oki-4N strain was cultured for growth and cultured under salt stress for 6 days (in A-3 medium containing 0.5 M NaCl, under strong light conditions), and salt-stressed algal cells were collected. The basic techniques for the culture and experiment were the same as those described in Experimental Example 2.
Total RNA was extracted from the collected cells using an RNA extraction reagent (Trizol reagent, Roche), and Poly(A)+ mRNA was isolated using a cDNA library creation kit (Smart-Infusion PCR cloning system, Clontech) to create a full-length cDNA library.
The obtained cDNA library was sequenced using a high-throughput next-generation sequencer (Illumina Hi Seq 2500 system, Illumina), and contig sequences were created from the fragment sequences using a high-speed sequence data analysis program (CLC Genomics Workbench, Qiagen), obtaining approximately 45,000 cDNA sequences.

(2) Determination of Amino Acid Sequence of Carotenoid-Binding Protein The partial amino acid sequence on the N-terminus of AstaP-pink1 and AstaP-pink2 purified in the above Experimental Examples was determined using an N-terminal amino acid sequencer (Shimadzu Corporation).
AstaP-orange2 purified in the above experimental example was subjected to SDS-PAGE, followed by trypsin treatment and purification treatment with a C18 column, and the mass of each peptide fragment after trypsin treatment was determined using an LC/MS/MS device (Orbitrap Q Exactive focus LC/MS/MS system, Thermo Scientific) equipped with a reversed-phase column (CAPCELL PAK C18 reversed-phase column, 150 × 2.0 mm id, 5 μm particle size, Shiseido). Analysis was performed from the MS/MS data of the peptide fragments using PMF analysis software (Peaks Studio 8.5, Bioinformatics Solutions), and the peptide sequence estimated from the theoretical value was estimated.

PCR primers were created based on the information of the partial amino acid sequences determined or estimated above, PCR amplification was performed using the cDNA library created in (1) above as a template, and the base sequences were determined using a capillary DNA sequencer. Then, from the sequence information and the sequence information of the cDNA library determined above, the base sequences of the cDNAs of the carotenoid-binding protein genes were determined, and the amino acid sequences encoded by the cDNAs were determined.
As a result, it was shown that the cDNAs of the genes encoding the AstaP-orange2, AstaP-pink1, and AstaP-pink2 proteins were cDNAs containing the nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively.
Furthermore, these amino acid sequences were shown to be the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively, as the primary amino acid sequences before processing.
Furthermore, for AstaP-orange2, AstaP-pink1, and AstaP-pink2, the amino acid sequences of the mature proteins from which the signal peptide was removed after processing were shown to be the amino acid sequences shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively. That is, it was shown that the carotenoid-binding proteins isolated above are proteins composed of these amino acid sequences as the amino acid sequences constituting their protein portions.

(3) Details of sequence information of carotenoid-binding protein Based on the amino acid sequence of the carotenoid-binding protein determined above, the characteristics obtained from the sequence information were analyzed. The bioinformatics analysis was performed using database information from NCBI and the like, GENETYX-MAC (Genetyx Corporation), and the like.

The amino acid sequence of AstaP-orange2 was shown to be composed of 402 amino acid residues as shown in SEQ ID NO: 4 as the primary amino acid sequence before processing ( FIG. 10 ). Here, the region composed of 19 amino acid residues shown in bold and underlined on the N-terminal side in FIG. 10 indicates a signal sequence, which is deleted in the mature protein (SEQ ID NO: 7: 383 amino acid residues) that functions as a carotenoid-binding protein.
The amino acid sequence was found to have H1 and H2 domains in the region enclosed by the box in the figure (bold italics), and the protein was found to belong to the Fasciclin family. AstaP-orange2 was found to have a domain structure duplication mutation in its ancestor gene, and was found to have a protein structure with two tandem pairs of H1 and H2 domains, instead of the usual pair.
Furthermore, the amino acid sequence contained a sequence motif for sugar chain modification (N-glycosylation site) in the region indicated by bold dashed underline, showing sequence characteristics that supported the results of the PAS staining described above.
Furthermore, the pI value, which indicates the isoelectric point of the functional protein after removal of the signal peptide, was estimated to be 3.6, and it was found to be an acidic protein.

The amino acid sequence of AstaP-pink1 was shown to be composed of 191 amino acid residues as shown in SEQ ID NO:5 as the primary amino acid sequence before processing (FIG. 11A). Here, the region composed of 24 amino acid residues shown in bold and underlined on the N-terminal side of FIG. 11A indicates a signal sequence, which is deleted in the mature protein (SEQ ID NO:8: 167 amino acid residues) that functions as a carotenoid-binding protein.
The amino acid sequence was found to contain H1 and H2 domains in the region enclosed by the box in the figure (bold italics), and the protein was found to belong to the Fasciclin family.
Furthermore, the amino acid sequence did not contain any sequence motif for sugar chain modification (N-glycosylation site), showing sequence characteristics that supported the results of the PAS staining described above.
Furthermore, the pI value, which indicates the isoelectric point of the functional protein after removal of the signal peptide, was estimated to be 4.7, and it was found to be an acidic protein.

The amino acid sequence of AstaP-pink2 was shown to be composed of 191 amino acid residues as shown in SEQ ID NO:6 as the primary amino acid sequence before processing (FIG. 11B). Here, the region composed of 24 amino acid residues shown in bold and underlined on the N-terminal side of FIG. 11B indicates a signal sequence, which is deleted in the mature protein (SEQ ID NO:9: 167 amino acid residues) that functions as a carotenoid-binding protein.
The amino acid sequence was found to contain H1 and H2 domains in the region enclosed by the box in the figure (bold italics), and the protein was found to belong to the Fasciclin family.
Furthermore, the amino acid sequence did not contain any sequence motif for sugar chain modification (N-glycosylation site), showing sequence characteristics that supported the results of the PAS staining described above.
Furthermore, the pI value, which indicates the isoelectric point of the functional protein after removal of the signal peptide, was estimated to be 3.8, and it was found to be an acidic protein.

(4) Gene phylogenetic tree Based on the amino acid sequence information determined above, the amino acid sequences of genes belonging to the fasciclin family of other organisms registered in the NCBI database were collected, a data set of alignable regions was created, and a gene phylogenetic tree was created by the neighbor-joining method. The phylogenetic analysis was performed in the same manner as in Experimental Example 1, except that amino acid sequences were used instead of base sequences.
In this phylogenetic analysis, in addition to AstaP-orange2, AstaP-pink1, and AstaP-pink2 as OTUs, the sequences of Marchantia polymorpha (Bryophyte Liverwort), Chlorella (Chlorophyceae), Monoraphidium (Chlorophyceae), Scutellaria (Chlorophyceae: a different species from Oki-4N), Coelastrella genus (Chlorophyceae), Botryococcus (Chlorophyceae), Coccomyxa (Chlorophyceae), Chrysochromulina (Haptophyceae), Fragilariopsis (Diatoms), Phaeodactylum (Diatoms), and Galdieria (Rhodophyceae) were added to the dataset and analyzed. The results are shown in FIG. 12.

As a result, it was shown that AstaP-orange2 of the Oki-4N strain (genus Scenedesmus) forms a clade with AstaP-orange1 (SEQ ID NO: 10: prior art) from the KI-4 strain, a microalga of the genus Coelastrella (Chlorophyceae), previously reported by the present inventor, and that the two genes (proteins) are homologous to each other.
On the other hand, AstaP-pink1 and AstaP-pink2 of the Oki-4N strain (genus Scenedesmus) formed a clade with each other, and it was shown that the genes (proteins) were highly homologous with extremely similar sequences in terms of branch length. In an alignment comparison of the primary amino acid sequences of both, the primary amino acid sequences of AstaP-pink1 and AstaP-pink2 showed a high sequence identity of 89% (170/191 amino acid residues).
In addition, in another species of the same genus Scenedesmus, there was one sequence (SEQ ID NO: 11: ACB06751) that showed homology to AstaP in the database, but in terms of the branch length, the sequence was not considered to have a high sequence homology with AstaP-pink1 and AstaP-pink2. Furthermore, the registered sequence was only sequence information, and its function as a carotenoid-binding protein was not disclosed.
Furthermore, AstaP-pink1 and AstaP-pink2 only partially show homology with AstaP-orange2 and AstaP-orange1 from the Ki-4 strain (SEQ ID NO: 10: prior art), and in terms of the topology of the phylogenetic tree, they are recognized to be in a paralogous relationship. The overall sequence identity of AstaP-pink1 and AstaP-pink2 with AstaP-orange1 is only 40% and 42%, respectively, and it was recognized that these sequence differences result in large differences in functional properties such as sugar chain modification patterns, color development properties, and color tones.

These results indicate that AstaP-orange2 is a protein that shows homology to AstaP-orange1 from the Ki-4 strain, a microalga of the genus Coelastrella (Chlorophyceae), while AstaP-pink1 and AstaP-pink2 are carotenoid-binding proteins with novel sequence characteristics that have not been reported in any other studies, including the Ki-4 strain.

(5) Primary Structure of Protein With respect to the sequence characteristics clarified above, the primary structure of the carotenoid-binding protein is summarized and shown in FIG. 13. For comparison, AstaP-orange1 (prior art) from the Ki-4 strain, a microalga of the genus Coelastrella (Chlorophyceae), is also shown in the same figure. In the figure, "sig" indicates a signal peptide, "H1" indicates the H1 domain, "H2" indicates the H2 domain, and "○" indicates a glycosylation site.

[Experimental Example 5] "Expression Pattern of Carotenoid Protein"
From the viewpoint of efficient production of the above carotenoid protein, the expression pattern of the carotenoid protein in the Oki-4N strain was analyzed.

(1) Gene Expression Analysis Northern hybridization analysis was carried out to examine the expression pattern of the mRNA of the gene encoding the carotenoid-binding protein.
The isolated Oki-4N strain was pre-cultured and cultured under salt stress for 4 days (in 0.5 M NaCl-containing A3-liquid medium, under strong light conditions), and the salt-stressed algal cells were collected and then total RNA was extracted. The basic method of the culture and experiment was the same as that described in Experimental Example 4.
The extracted total RNA (10 μg) was electrophoresed on an agarose gel and transferred onto a nylon membrane (Hybond N+ Nylon membrane, manufactured by GE Healthcare Japan, Ltd.) by the capillary blot method. A DNA fragment of a gene encoding a carotenoid-binding protein radioactively labeled with ³² P was applied as a probe to the membrane, and a hybridization reaction was carried out at 60° C. The membrane was washed and signal detection was carried out. PCR amplified fragments of cDNA of AstaP-pink1 and AstaP-orange2 were used as the probe DNA fragment. The results are shown in FIG. 14.

As a result, it was shown that the expression of AstaP-pink1 mRNA was detected one day after the start of salt stress culture, and the expression level increased two days after, and particularly after, four days. In addition, the expression of AstaP-orange2 mRNA was also detected one day after the start of salt stress culture, and the expression level increased four days after.
These results showed that the expression of the gene encoding the carotenoid-binding protein was increased by the salt stress culture and increased with the passage of culture time, indicating that the intracellular production of the carotenoid-binding protein was increased by the salt stress culture and increased with the passage of culture time.
Although the results for AstaP-pink2 are not shown in this example, considering that the AstaP-pink2 protein was isolated and purified in the salt stress culture in Experimental Examples 2 and 3, it was presumed that the expression pattern of the AstaP-pink2 gene was also increased and induced by salt stress culture, similar to that of the AstaP-pink1 gene.

(2) Changes in the Expression Pattern of Pigment Proteins When Salt Concentration is Changed The expression pattern of the above-mentioned carotenoid proteins was examined by examining the effect of salt stress culture with different salt concentrations.
The isolated Oki-4N strain was subjected to growth culture and salt stress culture for 2 weeks (A-3 liquid medium containing 0.25 M NaCl or 0.5 M NaCl, strong light), and the salt-stressed algal cells were collected, after which the water-soluble pigment fraction was separated.
The obtained water-soluble pigment fraction was analyzed using an HPLC-photodiode array detector (Hitachi, Ltd.) In the photodiode array detection, the relative signal intensity of each peak having absorbance in the visible light wavelength range (around 400 to 800 nm) was detected for the fractions separated by HPLC according to molecular weight.
The basic techniques for the culture and experiment were similar to those described in Experimental Example 2. The results are shown in FIG.

As a result, it was confirmed that peak 2 containing AstaP-orange2 and peak 3 containing AstaP-pink1 and AstaP-pink2 were detected in the water-soluble pigment fraction (water-soluble pigment fraction from cells) of cells cultured under salt stress of either 0.25 M NaCl or 0.5 M NaCl.
Here, when the culture was performed under a salt stress of 0.25 M, the expression level of peak 2 was high and the expression level of peak 3 was low. On the other hand, when the culture was performed under a salt stress of 0.5 M, the expression level of peak 2 was low and the expression level of peak 3 was high.
These results indicate that in order to increase the expression level of AstaP-orange2, it is desirable to set the NaCl concentration in salt stress culture to a low level, and that efficient pigment protein production is possible at 0.25 M.
On the other hand, in order to increase the expression levels of AstaP-pink1 and AstaP-pink2, it is desirable to set the NaCl concentration in salt stress culture to a high level, and it was shown that efficient pigment protein production is possible at 0.5 M.

(3) Mode of intracellular localization The cells cultured under salt stress (0.5 M NaCl-containing A-3 liquid medium, strong light) for 2 weeks as described in (2) above were observed using a confocal fluorescent microscope (Nikon Confocal Fluorescent Microscope). The microscopic observation was performed by obtaining a differential interference image (Nomarski) for observing the internal structure of the cells, a red fluorescent image (Red Fluorescence) at an excitation light of 630 nm/fluorescence of 685 nm for detecting the localization of chloroplasts, and a green fluorescent image (Green Fluorescence) at an excitation light of 480 nm/fluorescence of 530 nm for detecting the localization of carotenoid proteins (pigment proteins). In addition, overlapping images were synthesized using image analysis software (Nikon FluoView FV3000 software) for each photograph. The results are shown in FIG. 16.

As a result, it was shown that the green fluorescence, which indicates the presence of carotenoid proteins, was detected localized in organelles presumed to be vacuoles or endoplasmic reticulum. These results demonstrated that AstaP-orange2, AstaP-pink1, and AstaP-pink2 are water-soluble proteins that are mostly or at least partially localized in organelles presumed to be vacuoles or endoplasmic reticulum and accumulate within the cell.

[Summary of characteristics of carotenoid proteins]
As shown in the above experimental examples, it was shown that by culturing the new unicellular algae Oki-4N strain belonging to the genus Ligustrum under salt stress conditions, the expression of novel carotenoid-binding proteins AstaP-orange2, AstaP-pink1, and AstaP-pink2 was induced.
Most of these carotenoid-binding proteins exist as carotenoid proteins (pigment proteins) bound to astaxanthin, and they have been found to be proteins that enable astaxanthin, which is normally insoluble in water, to be used as a water-soluble pigment.
Here, the carotenoid protein of AstaP-orange2 exhibits a yellowish orange color when dissolved in water, similar to conventional astaxanthin and the prior art AstaP-orange1 of microalgae of the genus Coelastrella (Chlorophyceae), whereas the carotenoid proteins of AstaP-pink1 and AstaP-pink2 exhibited a pinkish red color when dissolved in water. In this regard, it was demonstrated that the carotenoid proteins of AstaP-pink1 and AstaP-pink2 can be used as novel bright red pigments not found in conventional astaxanthin.

1. Peak 1 in HPLC-photodiode array analysis from the water-soluble fraction
2. Peak 2 in HPLC-photodiode array analysis from the water-soluble fraction
3. Peak 3 in HPLC-photodiode array analysis from the water-soluble fraction

M. Marker O. AstaP-orange2
P1. AstaP-pink1
P2. AstaP-pink2
C. Control

Ax. Astaxanthin Ad. Adonixanthin Lt. Lutein Ca. Canthaxanthin

21. Organelles presumed to be vacuoles or endoplasmic reticulum 22. Chloroplasts

FERM P-22374

Claims (9)

A carotenoid binding protein,
( Sequence characteristics ) ( i ) comprising an amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 9, or ( ii ) comprising an amino acid sequence that shows 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 9 ,
( Functional feature 1 ) The carotenoid binding protein has a function of binding to astaxanthin to form a carotenoid protein, and
( Functional characteristic 2 ) When the carotenoid binding protein binds to astaxanthin to form a carotenoid protein, the carotenoid protein ( 1 ) exhibits the property of being soluble in water, ( 2 ) has singlet oxygen quenching activity when dissolved in water, and ( 3 ) exhibits a red color when dissolved in water .
A carotenoid-binding protein having the above sequence characteristics and the above functional characteristics 1 and 2 . The carotenoid-binding protein according to claim 1 , wherein the amino acid sequence of ( ii ) is an amino acid sequence that shows 95 % or more sequence identity to the amino acid sequence shown in SEQ ID NO:8 or SEQ ID NO:9. The carotenoid-binding protein according to claim 1 or 2, which, when an aqueous solution is prepared by dissolving the carotenoid protein according to ( Functional Feature 2 ) in pure water, exhibits an absorption spectrum with a maximum absorption wavelength of 495 to 540 nm. A carotenoid-binding protein having the characteristics described in ( A ) below:
( A ) The protein component is composed of the amino acid sequence shown in SEQ ID NO:8 or SEQ ID NO:9. A water-soluble carotenoid protein comprising the carotenoid-binding protein according to any one of claims 1 to 4 bound to a carotenoid. The water-soluble carotenoid protein according to claim 5, wherein the water-soluble carotenoid protein comprises, as a molecular group, a carotenoid protein bound to astaxanthin as a main molecule. A method for producing a water-soluble carotenoid protein, comprising the steps of:
The production method, comprising the step of mixing the carotenoid binding protein according to any one of claims 1 to 4 with a carotenoid. An astaxanthin-containing composition comprising the water-soluble carotenoid protein according to claim 5 or 6. A pigment composition, food or drink, cosmetic product, medicine, quasi-drug, daily hygiene product, feed, or reagent containing astaxanthin, which contains the water-soluble carotenoid protein according to claim 5 or 6.

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