JP4038573B2 - Transformation promoter for larval coral and method for producing the same - Google Patents

Description

The present invention relates to a transformation promoter effective for promoting transformation of larval coral larvae and a method for producing the same.

  Algae is a general term for a group of plants that perform aquatic photosynthesis, and includes a single cell having a diameter of about 0.8 μm to a large size having a length of 60 to 90 m or more. These algae are classified into 9 types of green algae plants, cyanobacteria plants, green caterpillar plants, golden algae plants, crypto-algae plants, flame-colored algae plants, green whip algae plants, brown algae plants and red algae plants. Characterized by composition, chemical composition of storage materials, number and location of flagella and physicochemical properties, etc., they are distinguished from each other.

For example, all algae contain chlorophyll a, carotene and xanthophylls, but green algae plants and green caterpillar plants additionally chlorophyll b, and brown algae plants and most flaming algae plants and golden plants contain chlorophyll c. Red algae plants contain chlorophyll d.
In addition, cyanobacteria and red algae plants contain phycopyrine pigments, but echinenone and myxoxanthophylls therein are peculiar to cyanobacteria plants, and red algae plants contain red algae starch similar to amylopectin.

In addition, among the components unique to these algae, there are substances exhibiting special physiological activities, and these are extracted and used for various purposes.
As for red algae plants, it has been reported that some of the substances derived from them have an action of promoting the settlement or transformation of marine invertebrate floating larvae.

  For example, powders of the red algae Corallinees, Corallinaceae, Hydrolithon, Hydrolythone reinbolnei, and the genus Pseudomonas It has been reported that seaweed powder promotes the growth or transformation of larvae of scleracinans (Acroporidae) corals (see Non-Patent Document 1).

  Further, a low molecular weight sulfated glycosaminoglycan having a molecular weight of 5 to 10 kilodaltons, which is purified from Corallinaceae, Corallinaceae, Hydrolithon reinboldii, is C It has also been clarified that there is an activity of promoting the growth or transformation of larvae of the genus Agariciidae (Agaricidae) (see Non-Patent Document 1).

Further, Benimotsuna coralline th (Corallinales) coralline family (Corallinaceae) Kobuishimo genus (Hydrolithon) Kobuishimo (Hydrolithon reinboldii) low molecular weight sulfated glycosaminoglycan was following molecular weight of 14,000 daltons purified from the, coral acids (S cleractinians ) It has also been clarified that there is an activity of promoting the growth or transformation of larvae of the genus Agariciidae, Agaricia humilis (see Non-Patent Document 2).

  By the way, seaweeds belonging to the order of red algae coral species such as the seaweeds of the genus Kobuisimo are generally slower in growth than large seaweeds of red algae plants and are difficult to produce in large-scale culture on land. Industrially mass-produces metamorphic promoters that promote the larval epithelial or metamorphic activity of larvae of the genus Acrocoridae, Agariciidae, and Agaricia humilis Is inappropriate.

  Similarly, because of its slow growth and difficulty in culturing in large quantities, larvae of larvae (Scleractinians) have been promoted with a transformation promoter having an activity of promoting the growth or transformation of seaweeds belonging to the order of red seaweeds. It is unsuitable to use and mass-produce. Therefore, for the production of transformation promoters such as Acroporidae coral and Agaricidae Agaricia humilis, it is preferable to use large seaweed that can be placed upright as a raw material. .

  As described above, seaweeds belonging to the genus Pegarsonalees (Pigsonneliaaceae) and the seaweeds belonging to the genus Peyssonnelia sp. Are known to exhibit metamorphosis or epithelial promoting effects on some coral larvae. However, this is a flat red algae that is generally slower in growth than a seaweed that can be placed upright, i.e., a seaweed that grows upright and has a large culture area. Is disadvantageous.

  Therefore, in order to obtain a transformation promoter having larval growth promoting or transformation promoting activity of the larvae of the reed corals (Scleractinians), the large seaweed Gigartinales (Gygartinales) Iwanokawa family (Peissoneliaceae) .) Seaweed is inappropriate.

  Moreover, seaweeds belonging to the order of the red coral class Coraloptera such as the seaweeds of the genus Kobuisimo and shell-like red algae such as seaweeds belonging to the genus Pegarsonaleaceae and the seaweed belonging to the genus Peyssonelia sp. Therefore, when these seaweeds are cultured on land, the substrate to which the seaweeds are attached is likely to be contaminated with other organisms and the like, so that it is difficult to continue the culture.

  In addition, the seaweeds of the genus Corallineaceae (Callolinaceae), Lithothamnium sp., The genus Corallinaceae, the genus Lithosp. (Hildenbrandiales) A member of the genus Hildenbrandiacee (Hildenbrandia sp.), Which is derived from shell-like red algae such as seaweeds, can improve the growth of Gastropoda araseis larvae. It has been reported (see Non-Patent Document 3), and the red seaweed large seaweed Oxenaceae (Ban iales) A component with absorption in the visible region 562 nm purified from the seaweed of Porphyra sp. promotes the epithelialization or transformation of Gastropoda radish larvae. (See Non-Patent Document 4).

  By the way, in general, seaweed that does not adhere to the substrate, or adheres to the substrate only with an attachment device corresponding to a small part of the alga body, and grows upright when the container is contaminated in terrestrial culture. The culture can be continued by transferring the upright portion or the tip of the upright portion to another container. Therefore, in order to produce a transformation accelerator by terrestrial culture, red algae coralid seaweeds and shell-like red algae are unsuitable, and it is preferable to use seaweeds that grow in an upright landing state, particularly monoalgal cultures.

  Here, the monoalgal culture strain is an algal strain obtained by treating marine algae and purifying it to one kind of algae, and is used for indoor cultivation of marine algae. This monoalgal culture can be prepared by, for example, isolating spores from mature algae and culturing them in artificial seawater, sterilized seawater, or artificial seawater.

  In general, natural marine algae are rich in surface epiphytes and endophytic parasites (endophytes). These epiphytic plants and endophytic parasites (1) make it difficult to continuously cultivate seaweed on land, and (2) high purity of transformation promoters in the purification process of transformation promoters derived from marine algae. May hinder. Therefore, by isolating spores from matured marine algae and culturing them in artificial seawater or sterilized seawater, we prepared monoalgae cultures, so that epiphytes and internal plants contained in natural marine algae The amount of sex parasites can be effectively reduced.

  For these reasons, there has been a demand for a method for efficiently producing a transformation promoter, particularly a transformation promoter for invertebrate larvae, by culturing a large amount on land using a fast growing red algae plant. So far, no such method has been known.

“Biol. Bullet”, 1996, Vol. 191, p. 149-154 “Biological Bulletin”, 1991, Vol. 181, p. 104-122 “Journal Experimental Marine Biology and Ecology” (J. Exp. Mar. Biol. Ecol.), 1984, Vol. 75, p. 191-215 “Hydrobiology”, 1984, 116/117, p. 155-158

The present invention has been made in order to solve the problem of providing a novel transformation promoter for larval coral larvae that can be efficiently produced by mass culture on land.

  The present inventors have conducted various studies on the physiological activity of components extracted from red algae plants, and as a result, extracted from large seaweeds belonging to red algae plants that can be placed upright, in other words, from large seaweeds other than shell-shaped red algae. The sulfated polysaccharide component, the sulfated glycosaminoglycan component, or the sulfated proteoglycan component exhibits metamorphosis promoting action and implantation promoting action on invertebrate larvae such as coral corals, and these components are It was found that the extract was concentrated to a specific fraction of the extract from and separated from this, and the present invention was made based on this finding.

That is, the present invention relates to a saline solution of a large-sized seaweed that can stand upright belonging to the order of Rhodophyta, Rhodophyceae, Gracilariaes, Gigartinales, or Ceramiales. Among the fractions extracted with an aqueous salt solution selected from phosphate buffer, Tris-HCl buffer and sodium chloride-containing phosphate buffer, a fraction having a molecular weight of 20,000 to 90,000 or a wavelength of 480 to 480 transformation accelerator coral such larvae comprising a fraction having an absorption maximum in 570nm region as an active ingredient, and red algae plants Gate (Rhodophyta) Benimotsuna (Rhodophyceae) Gracilaria th (Gracilariales), Suginori th (Gigartinales) the Is a large seaweed belonging to the order of Ceramiales, which can be placed upright and extracted with an aqueous salt solution selected from physiological saline, phosphate buffer, Tris-HCl buffer and sodium chloride-containing phosphate buffer. Then, first, ammonium sulfate was added to the extract to a final concentration of 30 to 40% by mass to perform salting out in the first stage to remove precipitated contaminants, and the extract was further added to a final concentration of 70% by mass. Ammonium sulfate was added until the concentration reached about 2%, salting out in the second stage, and the fraction of the crude active ingredient obtained as a precipitate was collected, followed by gel filtration chromatography to obtain a fraction having a molecular weight of 20,000 to 90,000. or the partial fractionation or fractionated fractions having an absorption maximum in the region of the chromatography to wavelength 480~570nm min, the coral such larvae There is provided a method for producing a transformation accelerator coral such larvae, characterized in that collecting the fractions showing transformation activity.

  Next, the present invention will be described in more detail. The classification of seaweed was based on [Tadao Yoshida, “New Japan Seaweed Magazine”, Uchida Otsukuru, 1998].

  The transformation promoter of the present invention comprises an extract from a large-sized seaweed that can be placed upright, belonging to the order of the red algae of the red algae plant genus Ogonori, Suginotidae, or Igidae. “Implantable” means that it can be grown on a solid object such as a rock with an applicator and kept upright, and large seaweed means seaweed with a leaf-like part length of 5 mm or more. means.

  As the raw material of the present invention, large seaweeds belonging to the order of Gracilariaes, Gigartinales, or Ceramiales are preferable.

  Examples of such Gracilariaes seaweeds include Gracilariaceae seagrass, Gracilariaceae, Gracilariaceae, Examples include the genus (Gracilaria chorda) and the like.

  Examples of seaweeds belonging to the order of Gigartinales include Kallymeriaceae (Callophyllia sp.), Kallymenaceae (Callomyaceae) Rhizophyllidaceae, P. sp., Rhizophyllidaceae, P., P., J., and M. cereal.

Furthermore, examples of seaweeds belonging to the order of Ceramiales include Laodomelaceae seaweeds, Rhodomelaceae Sozoa (Laurenciae), and the like.
As a raw material used in the present invention, naturally grown seaweed may be used, but a monoalgae culture strain is preferable.

The transformation promoter of the present invention extracts, for example, a large-sized seaweed that can be placed upright, belonging to the aforementioned red algae class, with a salt-containing aqueous solution, and ammonium sulfate is first added to this extract until a final concentration of 30 to 40% by mass The first stage of salting out is carried out to remove the precipitated contaminants, and ammonium sulfate is further added to the extract to a final concentration of about 70% by mass, followed by the second stage of salting out to obtain a precipitate. The resulting crude active ingredient fraction is fractionated, and then fractionated with a molecular weight of 20,000 or more and 90,000 or less by gel filtration chromatography, or the precipitate is chromatographed to obtain a wavelength of 480 to 550 nm. It can be produced by fractionating a fraction having an absorption maximum in the region and collecting fractions that show metamorphosis-promoting activity against invertebrate larvae such as coral corals. That.
The salt- containing aqueous solution used at this time is selected from physiological saline, phosphate buffer, Tris-HCl buffer, and sodium chloride-containing phosphate buffer .

  In order to suitably carry out this method, the following raw materials are extracted from the above-mentioned raw materials (a) a water-soluble fraction extraction step, (b) a crude active ingredient fraction fractionation step, and (c) a transformation accelerator purification step. Can be applied sequentially.

  In this case, in the step (a), a salt-containing aqueous solution, for example, sodium chloride-containing phosphate buffer is added to the raw material and homogenized, and then centrifuged to obtain a crude extract as a supernatant.

  Next, in the step (b), ammonium sulfate is first added to the extract obtained in the step (a) so that the final concentration becomes a saturated solution of about 30 to 40% by mass, and the first stage salting out is performed. And the produced precipitate is removed by centrifugation. By this operation, impurities such as pigment are removed as a precipitate fraction. Next, the supernatant is centrifuged, and ammonium sulfate is added to the saturated solution with a final concentration of about 70% by mass for salting out in the second stage, and the resulting precipitate is separated by centrifugation. The precipitated fraction is redissolved with a buffer such as sodium chloride-containing phosphate buffer to obtain a crude active ingredient fraction.

  Next, in the step (c), the crude active ingredient fraction obtained in the step (b) is dialyzed against a buffer solution such as a sodium chloride-containing phosphate buffer and then subjected to gel filtration chromatography to a molecular weight of 20, Fractions of 000 to 90,000 are fractionated, and fractions showing activity such as metamorphosis promotion for invertebrate larvae such as coral corals are collected. If necessary, the purified product can be obtained by further separating the components by chromatography. In this case, it is preferable to use ion exchange chromatography, gel filtration chromatography, hydrophobic interaction chromatography, or a combination thereof as the chromatography used in the final stage.

  The fraction having a molecular weight of 20,000 or more and 90,000 or less here means that the result of calculating the molecular weight of the eluted fraction using a globular protein as a standard molecular weight substance in gel filtration chromatography is 20,000 or more and 90. Refers to the fraction corresponding to a molecular weight of 1,000 or less.

  In the step (c), the crude active ingredient fraction obtained in the step (b) is dialyzed against a buffer solution such as a sodium chloride-containing phosphate buffer and chromatographed to obtain a wavelength of 480 to 570 nm. It is also possible to fractionate a fraction exhibiting an absorption maximum in the visible region and collect a fraction containing a purified red dye. In this case also, if necessary, the purified product can be obtained by further separating the components by chromatography. In this case, the chromatography used in the final stage is preferably ion exchange chromatography, gel filtration chromatography, hydrophobic interaction chromatography, or a combination thereof.

  The fraction showing the absorption maximum in the above-mentioned region of 480 to 570 nm is collected, for example, by separating the eluate from the chromatography column over time as a small amount, preferably a fraction less than the column volume, and 480 for each fraction. It can be carried out by measuring the absorbance in the visible region up to 570 nm and collecting the fraction showing the maximum absorption in this region.

  The substance recovered from the fraction thus obtained is a novel substance not described in the literature, characterized by having a physiological activity of promoting settlement or metastasis to the coral larvae, which is a sulfated polysaccharide component or It is considered that the main component is either a sulfated glycosaminoglycan component, a sulfated proteoglycan component or a physiologically active dye.

The transformation promoter of the present invention is a novel substance derived from a large seaweed belonging to the order of the red algae Ogonori, Sugiori, or Hygiidae, has a transformation promoting activity against larval corals , and is established on the same larvae. Has promoting activity. Moreover, since this substance is derived from a red algal plant that can be placed upright, it can be produced in large quantities by culturing in large quantities on land.

Next, the best mode for carrying out the present invention will be described by way of examples, but the present invention is not limited thereto.
The “transformation rate” in each example means the ratio (%) of the total number of samples that have reached the stage (single polyp stage) where the coral larvae have grown on the base layer and formed a radial skeleton. To do.

(I) Extraction process of water-soluble fraction As a raw material, using the order of Gracilariaes, Gracilariaceae, Gracilaria, Gracilaria chorda (produced in Yoshinogawa estuary, Tokushima Prefecture) After washing with an aqueous solution, it was dried in the sun to obtain a dried product. Next, after adding 700 ml of 0.15 M sodium chloride-containing 100 mM phosphate buffer (pH 6.9) to 100 g of this dried product and homogenizing, the homogenized solution was allowed to stand at 4 ° C. for 6 hours, then centrifuged, As a result, a crude extract was obtained.

(B) Separation step of the crude active ingredient fraction Next, ammonium sulfate was added to the crude extract so as to obtain a saturated solution having a final concentration of 35% by mass, and the first stage salting out was performed. After completion of the addition of ammonium sulfate, the mixture was allowed to stand at 4 ° C. for 1 hour, and the produced precipitate was removed by centrifugation. By this operation, contaminants such as pigment were removed as a precipitate fraction. Next, ammonium sulfate was added to the supernatant obtained by centrifugation so as to be a saturated solution having a final concentration of 70% by mass, and the mixture was allowed to stand at 4 ° C. overnight, and then the produced precipitate was separated by centrifugation. The fractionated precipitate fraction was redissolved with 100 mM phosphate buffer (pH 6.9) containing 0.15 M sodium chloride to obtain a crude active ingredient fraction.

(C) Purification step of the crude active ingredient fraction Next, the crude active ingredient fraction thus obtained was dialyzed against 100 mM phosphate buffer (pH 6.9) containing 0.15 M sodium chloride, After removing the insoluble contaminant protein by centrifugation, a fraction having a molecular weight of 20,000 to 90,000 was fractionated by gel filtration chromatography to obtain a purified product. As a result of analysis of carbohydrates, sulfate groups, and proteins in the obtained purified product, the hexose content is about 6 mg (by the phenol-sulfuric acid method) and the sulfate group content is about 1 when compared in purified products of the same mass. 0.5 mg (by rhodizonate method), protein content was 0.2 mg (by Lowry method) or less. From the results of chemical analysis, this purified product was estimated to be a sulfated polysaccharide component, a sulfated glycosaminoglycan component, or a sulfated proteoglycan component.

The purified product was measured for metamorphosis-promoting activity against larvae ( S cleactinians) Acroporidae Acroporia hyacinthus larvae.

  Since most of the Coralidae corals, such as Kashihadamidai, are hermaphrodite and gamete-releasing type reproductive ecology, these corals are a bundle of eggs (spermbundles) encased in multiple eggs with sperm and buoyancy. discharge. Collection of egg coral bundle (egg-spermbundle) and fertilization were performed as follows.

A mature group of four swordfish was collected at a depth of 5 to 10 m off the Pacific Ocean off the Yokonami Peninsula, Kochi Prefecture. It was stored in a water tank set up in a cold and cold laboratory until just before the release of eggs.
Next, kushihamidorii was divided into individual tanks of filter sterilized seawater for each group. As the filter sterilized seawater, seawater first filtered with a 1 μm glass filter and finely filtered with a 0.22 μm Millipore filter was used. After the start of egg laying (bundle release), the gametes released from the group 1 were collected and added to filter sterilized seawater. The eggs and sperm were transferred to a container containing filtered sterilized seawater, and cross-fertilized with eggs or sperm separated from other colonies. The larvae of the resulting swordfish were stored at 25 ° C. in a container containing filter sterilized seawater.

  After maintaining the larvae of the bark beetle at 25 ° C. for 8 days, the larvae of 10 individuals are placed in a 20 ml capacity polystyrene cup containing 10 ml of filter-sterilized seawater and allowed to stand, and the transformation rate of the coral larvae after 72 hours is obtained. It was. As a sample at this time, a mixture of 1 ml of the purified product and 9 ml of filter sterilized seawater was used, and a blank of 10 ml of filter sterilized seawater was used as a blank. The results are shown in Table 1.

Comparative Example 1
As a raw material, sterilized the seaweed with 3 sterilized seawater using the brown algae Dictyotales (Dictyoaceae) Lobophora (Lobophora variegata) collected between 5 and 10 m in depth from the Pacific Ocean off the Yokonami Peninsula, Kochi Prefecture. Washed once and lightly removed water on the filter paper. Taking 1 g of this wet grass mass, 10 ml of filter-sterilized seawater was added and ground at 4 ° C. using a mortar and pestle to prepare a brown algae suspension. Using this, in the same manner as in Example 1, the physiological activity on the larvae of Kushihamidorii was examined. The results are shown in Table 1.

  As is clear from Table 1, the purified product (a seaweed-derived sulfated polysaccharide component, sulfated glycosaminoglycan component or sulfated proteoglycan component) has an activity of promoting the transformation of sea coral larvae. Further, the filter sterilized seawater and the brown algae suspension were not recognized to promote the activity of the larval metamorphosis.

  As a raw material, instead of Gracilariaes, Gracilariaceae, Gracilaria, Gracilaria chorda (from the Yoshinogawa estuary, Tokushima Prefecture) (B) Steps were carried out to obtain a crude active ingredient fraction. Gigartinales (Rhizophyllidaceae) Naminohana (Portieria japonica) is the same inhabiting from 5 to 10 m in the Pacific Ocean off the Yokonami Peninsula in Kochi Prefecture. Used.

This crude active ingredient fraction is passed through gel filtration chromatography, and fractions having a molecular weight of 20,000 or more and 90,000 or less are collected and concentrated to obtain a sulfated saccharide derived from naminohana (sulfated polysaccharide component or sulfated glucosaminoglycan). Component or sulfated proteoglycan component) fraction. Similarly coral such as in Example 1 Naminohana from sulfated saccharide fraction (S cleractinians) Midoriishi family of (Acroporidae) Kushihadamidoriishi (Acropora Hyacinthus) transformation promoting activity against larvae was measured. The transformation rates in the two experiments were 30% and 30%, and the average value was 30%.

  As a raw material, instead of Gracilariaes, Gracilariaceae, Gracilaria, and Gracilaria chorda (produced in the Yoshinogawa estuary, Tokushima Prefecture) Steps (a) and (b) were carried out to obtain a crude active ingredient fraction. The genus Gigartinales (Kallymeriaceae) and the genus Callophyllis (Callophyllicus japonica) are inhabited in the Pacific Ocean off the coast of Yokonami Peninsula, 5m to 10m. It was collected from and used.

This crude active ingredient fraction is passed through gel filtration chromatography, and fractions having a molecular weight of 20,000 or more and 90,000 or less are collected, concentrated, and sulfated saccharide (sulfated polysaccharide component or sulfated glucose) Saminoglycan component or sulfated proteoglycan component) fraction was obtained. In the same manner as in Example 1, the sulfate-promoting saccharide fraction derived from Spodoptera litura was measured for metastasis-promoting activity on larvae ( S cleractinians), Acropora hyacinthus larvae. The transformation rates in the two experiments were 20% and 30%, and the average value was 25%.

  As a raw material, instead of Gracilariaes, Gracilariaceae, Gracilaria, and Gracilaria chorda (produced in the Yoshinogawa estuary, Tokushima Prefecture), Laurenciaa 1 (B) Steps were carried out to obtain a crude active ingredient fraction. Ceramiales, Rhodomelaceae, Laurencia, and Laurencia pinnata are inhabited by the waters of Kushihadamiridi, which are collected from the waters of the Pacific Ocean off the coast of Yokonami Peninsula, Kochi Prefecture. Used.

This crude active ingredient fraction is passed through gel filtration chromatography, and fractions having a molecular weight of 20,000 or more and 90,000 or less are collected, concentrated, and honeysozo derived sulfated saccharide (sulfated polysaccharide component or sulfated glucosaminoglycan). Component or sulfated proteoglycan component) fraction. In the same manner as in Example 1, the haemozozo-derived sulfated carbohydrate fraction was measured for metamorphosis-promoting activity against larvae ( S cleactinians) Acroporidae (Acroporia hyacinthus) larvae. The transformation rates in the two experiments were 20% and 20%, and the average value was 20%.

  As a raw material, instead of Gracilariaes, Gracilariaceae, Gracilaria, Gracilaria chorda (produced in the Yoshinogawa estuary, Tokushima Prefecture) ) And (b) steps were performed to obtain a crude active ingredient fraction. Gigartinales (Serieriaceae), Eucheuma and Eucheuma serra were collected from the Pacific Ocean off Towashima Prefecture at a depth of 5 to 10 m and used for experiments.

This crude active ingredient fraction is passed through gel filtration chromatography, and fractions having a molecular weight of 20,000 or more and 90,000 or less are collected and concentrated, and the sucrose derived sulfated saccharide (sulfated polysaccharide component or sulfated glucosamino). Glycan component or sulfated proteoglycan component) fraction (molecular weight 20,000 or more) was obtained. In the same way as in Example 1, the metamorphosis promoting activity for larvae ( S cleactinians) and Acroporia hyacinthus larvae was measured for the sulfated saccharide fraction derived from Togekirinsai. The transformation rates in the two experiments were 20% and 20%, and the average value was 20%.

Comparative Example 2
In the fractionation step of the crude active ingredient fraction of Example 1 (b), instead of fractionation by two-stage salting out by addition of ammonium sulfate, fractionation with 50% by mass ethyl alcohol [“Phytochemistry”, 1988, 27, p. The crude fraction was obtained in the same manner as in Example 1 except that the process was carried out in the same manner as in Example 1. This crude fraction was passed through gel filtration chromatography, and fractions having a molecular weight of 20,000 to 90,000 were collected and concentrated to obtain an ethyl alcohol fraction. Similarly coral such as in Example 1 ethyl alcohol fractionated fraction (S cleractinians) Midoriishi family of (Acroporidae) Kushihadamidoriishi (Acropora Hyacinthus) transformation promoting activity against larvae was measured. As a result, no activity to promote metamorphosis was detected in the ethyl alcohol fraction.

Reference examples Thyroglobulin (mass average molecular weight 669,000), ferritin (mass average molecular weight 440,000), bovine serum albumin (mass average molecular weight 67,000), ovalbumin (mass) purchased from Amersham Biosciences as standard molecular weight proteins Using the average molecular weight 43,000) and ribonuclease A (mass average molecular weight 13,700), the relationship between the elution volume of gel filtration chromatography and the mass average molecular weight was determined.
The standard protein thus obtained and the elution volume in gel filtration chromatography were as follows: the elution volume of thyroglobulin was 6.56 ml, the elution volume of ferritin was 6.78 ml, the elution volume of bovine serum albumin was 7.51 ml, It was found that the elution volume of albumin was 7.91 ml and the elution volume of ribonuclease A was 9.05 ml.
From this result, it can be seen that the common logarithm Y of the mass average molecular weight and the elution volume X in gel filtration chromatography have the following relationship.
Y = 10.241118407-0.691137206X (I)
The correlation coefficient of this equation was −0.971.

Next, 0.1 ml of a sulfated carbohydrate component (sulfated polysaccharide component, sulfated glycosaminoglycan component or sulfated proteoglycan component) derived from a large seaweed of a red algae plant having transformation promoting activity is passed through a TSKgel G3000PWxl column, and the gel Eluted fractions were collected in 0.1 ml portions from the filtration chromatography column. The fraction having a total elution volume from the column of 0.05 to 0.15 ml was designated as fraction number 1. (Thus, the fraction of fraction number 50 fractionates the total elution volume from the column from 4.95 to 5.05 ml. The elution volume of this fraction was averaged to be 5.00 ml). Each collected elution fraction was subjected to sugar determination by the phenol-sulfuric acid method and calculated according to the elution volume and the formula (I).
Based on this result, the graph of the relationship between the elution volume in gel filtration chromatography and the sugar content of each fraction is shown in FIG.

As a result, the peak apex (elution volume: 7.90 ml) indicating the eluted fraction of the seaweed-derived sulfated carbohydrate component having transformation promoting activity corresponded to a molecular weight of 60,200.
Further, from the formula (I), the elution volume range of components having a molecular weight of 20,000 or more and 90,000 or less is 7.647 ml or more and 8.592 ml or less, and the peak of the peak of the seaweed-derived sulfated carbohydrate component having transformation promoting activity is obtained. It can be seen that an elution volume of 7.90 ml is included in the range.

(I) Extraction process of water-soluble fraction As a raw material, using the order of Gracilariaes, Gracilariaceae, Gracilaria, Gracilaria chorda (produced in Yoshinogawa estuary, Tokushima Prefecture) After washing with an aqueous solution, it was dried in the sun to obtain a dried product. Next, after adding 700 ml of 0.15 M sodium chloride-containing 100 mM phosphate buffer (pH 6.9) to 100 g of this dried product and homogenizing, the homogenized solution was allowed to stand at 4 ° C. for 6 hours, then centrifuged, As a result, a crude extract was obtained.

(B) Separation step of the crude active ingredient fraction Next, ammonium sulfate was added to the crude extract so as to obtain a saturated solution having a final concentration of 35% by mass, and the first stage salting out was performed. After completion of the addition of ammonium sulfate, the mixture was allowed to stand at 4 ° C. for 1 hour, and the produced precipitate was removed by centrifugation. By this operation, impurities such as a purple pigment were removed as a precipitate fraction. Next, ammonium sulfate was added to the supernatant obtained by centrifugation so as to be a saturated solution having a final concentration of 70% by mass, and the mixture was allowed to stand at 4 ° C. overnight, and then the produced precipitate was separated by centrifugation. The separated precipitate fraction was redissolved with 25 mM Tris-HCl buffer (pH 7.6) to obtain a crude active ingredient fraction.

(C) Step of purifying active ingredient Next, the crude active ingredient fraction obtained in this manner was dialyzed against 25 mM Tris-HCl buffer (pH 7.6), centrifuged, and insoluble contaminant protein. A re-dissolved active ingredient fraction from which was removed was prepared. A gel filtration column (inner diameter 2.6 cm, gel height 21.0 cm, gel volume about 120 ml) packed with 120 ml of this re-dissolved active ingredient fraction with a filler (trade name “DEAE Toyopearl 650S” manufactured by Tosoh Corporation) Used, ion exchange chromatography was performed by increasing the concentration of sodium chloride in the mobile phase from 0 to 0.21M. During this time, 10 ml elution fractions were collected from the ion exchange chromatography column, and the absorbance at 540 nm in the visible region was measured with a 10 mm cell. In this way, fraction Nos. 174 to 272 having an absorbance at a visible part of 540 nm of 0.01 or more were collected to obtain a purified product. FIG. 2 shows the relationship between this fraction and absorbance. As a result of measuring the absorption spectrum of this purified product, it was confirmed that it was a component having an absorption maximum in the visible region between 480 and 570 nm. FIG. 3 shows the absorption spectrum of fraction No. 220. The absorption maximum data of fraction No. 220 of the eluted fraction were 497 nm, 541 nm, and 564 nm in the visible region from 480 to 570 nm.

The mobile phase conditions of ion exchange chromatography in this example are as follows.
That is, after completion of the addition of the redissolved active ingredient fraction to the ion exchange column, the column was washed with 25 mM Tris-HCl buffer (pH 7.6) of about 10 times the column volume, and then the sodium chloride concentration in the buffer was adjusted. The red active ingredient adsorbed on the column was collected by linear increase from 0 to 0.21M. In this case, the total volume of the mobile phase when the sodium chloride concentration was increased from 0 to 0.21 M was about 13 times the column volume.

The purified product thus obtained was measured for metamorphosis-promoting activity against larvae ( S cleactinians) Acroporidae Acroporia hyacinthus larvae.

  Since most of the Coralidae corals, such as Kashihadamidai, are hermaphrodite and gamete-releasing type reproductive ecology, these corals are a bundle of eggs (spermbundles) encased in multiple eggs with sperm and buoyancy. discharge. In this experiment, a collection of egg coral bundles (egg-spermbundle) and fertilization were performed as follows.

  Four mature scorpions were collected at a depth of 5 m to 10 m off the Pacific Ocean off the Yokonami peninsula in Kochi Prefecture, and stored in a water tank set up in a laboratory cold and dark place until just before the scuttlefish was released. Next, kushihamidorii was divided into individual water tanks (containing filtered sterilized seawater) for each group. As the filter sterilized seawater, seawater first filtered with a 1 μm glass filter and finely filtered with a 0.22 μm Millipore filter was used. After the start of egg laying (bundle release), the gametes released from the group 1 were collected and added to filter sterilized seawater. The eggs and sperm were transferred to another container (contains filtered sterilized seawater) and cross-fertilized with eggs or sperm separated from other colonies. The larvae of the resulting swordfish were stored at 25 ° C. in a container containing filter sterilized seawater.

  After maintaining the larvae of the damselfly at 25 ° C. for 8 days, 10 larvae were placed in a 20 ml volume polystyrene cup containing 10 ml of filter-sterilized seawater and allowed to stand, and the number of metamorphosis of the coral larvae after 72 hours was obtained. It was.

  As a sample, a mixture of 1 ml of purified product and 9 ml of filter sterilized seawater was used, and 10 ml of filter sterilized seawater alone was used as a blank. The results are shown in Table 2.

Comparative Example 3
As a raw material, sterilized brown seaweed from the 5 to 10 m depth of Pacific Ocean off the Yokonami peninsula, Kochi Prefecture, using the algae (Dictyotales), Dictyotaceae, Lobophora variega After washing 3 times, the water was lightly removed on the filter paper. Taking 1 g of this wet grass mass, 10 ml of filter-sterilized seawater was added and ground at 4 ° C. using a mortar and pestle to prepare a brown algae suspension. The transformation rates of these samples were determined in the same manner as in Example 6, and the results are shown in Table 2.

  As is clear from Table 2, the purified product (a component having an absorption maximum in the visible region between 480 and 570 nm) has an activity of promoting metamorphosis of sea coral larvae. Moreover, the activity which promotes a coral larva metamorphosis was not recognized at all about filtration sterilized seawater and brown algae suspension.

  As a raw material, instead of Gracilariaes, Gracilariaceae, Gracilaria, Gracilaria chorda (from the Yoshinogawa estuary, Tokushima Prefecture) (B) Steps were carried out to obtain a crude active ingredient fraction. Gigartinales (Rhizophyllidaceae) Naminohana (Portieria japonica) is the same inhabiting from 5 to 10 m in the Pacific Ocean off the Yokonami Peninsula in Kochi Prefecture. Used.

This crude active ingredient fraction was subjected to ion exchange chromatography in the same manner as in Example 6, and a fraction having an absorbance at a visible part of 540 nm of 0.01 or more was collected to obtain a purified product as a transformation promoting component derived from Naminohana. In this refined product, the metamorphosis promoting activity on larvae ( S cleactinians) Acroporidae (Acroporia hyacinthus) larvae was measured in the same manner as in Example 6, and the transformation rates in the two experiments were 20% and 20%. The average value was 20%.

  As a raw material, instead of Gracilariaes, Gracilariaceae, Gracilaria chorda (from Yoshinogawa estuary, Tokushima Prefecture) Steps (a) and (b) were carried out to obtain a crude active ingredient fraction. The genus Gigartinales (Kallymeriaceae) and the genus Callophyllis (Callophyllicus japonica) are inhabited in the Pacific Ocean off the coast of Yokonami peninsula. It was collected from and used.

This crude active ingredient fraction was subjected to ion exchange chromatography in the same manner as in Example 6, and a fraction having an absorbance at a visible portion of 540 nm of 0.01 or more was collected, whereby a purified product was obtained as an active ingredient derived from Spodoptera litura. . In this refined product, the metamorphosis promoting activity on larvae ( S cleactinians) Acroporidae (Acroporia hyacinthus) larvae was measured in the same manner as in Example 6, and the transformation rates in the two experiments were 20% and 20%. The average value was 20%.

  As a raw material, instead of Gracilariaes, Gracilariaceae, Gracilaria, and Gracilaria chorda (produced in the Yoshinogawa estuary, Tokushima Prefecture), Laurenciaa 1 (B) Steps were carried out to obtain a crude active ingredient fraction. Ceramiales, Rhodomelaceae, Laurencia, and Laurencia pinnata are inhabited by the waters of Kushihamidorii between 5m and 10m in the Pacific water off Yokonami Peninsula, Kochi Prefecture. Used in the experiment.

This crude active ingredient fraction was subjected to ion exchange chromatography in the same manner as in Example 1, and a fraction having an absorbance at a visible part of 540 nm of 0.01 or more was collected to obtain a purified product as an active ingredient derived from honeysozo. As for the active ingredient derived from the honey beetle, the metastasis-promoting activity on the larvae ( S cleactinians), Acroporidae, Acroporia hyacinthus larvae was measured as 20% and 20 in the two experiments. The average value was 20%.

  As a raw material, instead of Gracilariaes, Gracilariaceae, Gracilaria, and Gracilaria chorda (produced in the Yoshinogawa estuary, Tokushima Prefecture) ) And (b) steps were performed to obtain a crude active ingredient fraction. Gigartinales (Serieriaceae) Eucheuma and Eucheuma serra were collected in the Pacific Ocean off Hiwasa, Tokushima Prefecture at a depth of 5 to 10 m for use in experiments.

This crude active ingredient fraction was subjected to ion exchange chromatography in the same manner as in Example 6, and a fraction having an absorbance at a visible part of 540 nm of 0.01 or more was collected to obtain a purified product as an active ingredient derived from stalks. In this refined product, the metamorphosis promoting activity on larvae ( S cleactinians) Acroporidae (Acroporia hyacinthus) larvae was measured in the same manner as in Example 6, and the transformation rates in the two experiments were 20% and 20%. The average value was 20%.

Comparative Example 4
In the fractionation step of the crude active ingredient fraction of Example 6 (b), instead of fractionation by salting out in two steps with ammonium sulfate addition, fractionation with 50% by mass ethyl alcohol [“Phytochemistry”, 1988, 27, p. The crude fraction was obtained in the same manner as in Example 6 except that the process was carried out in the same manner as in Example 6. As in Example 6, this crude fraction was measured for the metamorphosis promoting activity on larvae ( S cleractinians), Acroporidae, and Acroporia hyacinthus. No activity was detected.

Comparative Example 5
As a raw material, instead of Gracilariaes, Gracilariaceae, Gracilaria, Gracilaria chorda (from Tokushima Prefecture, Yoshinogawa estuary), a red seaweed large seaweed Using the genus seaweed (Porphyra sp.), The steps (i) and (b) of Example 6 were performed to obtain a crude red pigment fraction.

The crude red pigment fraction was subjected to ion exchange chromatography in the same manner as in Example 6, and the fraction having an absorbance at a visible part of 540 nm of 0.01 or more was collected to obtain a purified product as a red pigment component derived from the seaweed genus seaweed. Obtained. In this purified product, the metamorphosis promoting activity on larvae ( S cleactinians) Acroporidae (Acroporia hyacinthus) larvae was measured in the same manner as in Example 6, and the transformation rates in the two experiments were 0% and 0%. The average value was 0%.

  The transformation promoter of the present invention is used in, for example, the fishery industry, the medical field, and the biochemical industry, for example, coral larvae settlement promoters, coral larvae metamorphosis promoters, coral species preservatives, coral reef preservatives, and testing. Useful as a reagent.

The graph which shows the relationship between the elution volume in the gel filtration chromatography in a reference example, and the sugar content of each fraction. The graph which shows the light absorbency of 540 nm of visible parts measured with the cell of length 10mm about the elution fraction of the crude active ingredient fraction obtained in Example 6. FIG. The graph which shows the absorption spectrum of the fraction number 220 of the elution fraction of the crude active ingredient in Example 6. FIG.

Claims (3)

Rhodophyta, Rhodophyceae, Gracilariales, Gigartinales, or Ceramiales, a large seaweed that can be placed upright , physiological saline, phosphate buffer Among the fractions extracted with an aqueous salt solution selected from Tris-HCl buffer and sodium chloride-containing phosphate buffer , the absorption maximum in a fraction having a molecular weight of 20,000 to 90,000 or a wavelength of 480 to 570 nm A transformation promoter for larval coral larvae, comprising a fraction containing Rhodophyta plant Gate (Rhodophyta) Benimotsuna (Rhodophyceae) Gracilaria th (Gracilariales), the Suginori eyes (Gigartinales) or upright implantation possible large marine algae belonging to Igisu th (Ceramiales), saline, phosphate buffer The aqueous solution is extracted with an aqueous salt solution selected from Tris-HCl buffer and sodium chloride-containing phosphate buffer , and then ammonium sulfate is first added to the extract to a final concentration of 30 to 40% by mass. After removing the precipitated contaminants, ammonium sulfate is further added to the extract to a final concentration of about 70% by mass, and the second stage of salting out is performed to obtain a crude active ingredient obtained as a precipitate. Fractions are collected and then subjected to gel filtration chromatography to a molecular weight of 20,000 or more. 90,000 fractionated following fractions minute method of transformation accelerator stony coral such larvae of claim 1, wherein the collecting the fractions showing transformation promotion activity on the stony coral such larvae. Rhodophyta plant Gate (Rhodophyta) Benimotsuna (Rhodophyceae) Gracilaria th (Gracilariales), the Suginori eyes (Gigartinales) or upright implantation possible large marine algae belonging to Igisu th (Ceramiales), saline, phosphate buffer Extraction with an aqueous salt solution selected from Tris-HCl buffer and sodium chloride-containing phosphate buffer, and ammonium sulfate is first added to the resulting extract to a final concentration of 30 to 40% by mass. After salting out the eyes and removing the precipitated contaminants, further addition of ammonium sulfate to the extract to a final concentration of about 70% by mass, salting out in the second stage, crude activity obtained as a precipitate The component fraction is separated and then chromatographed to give a wavelength of 480-570n. Fractionated fractions having an absorption maximum in the region min, method for producing a transformation accelerator stony coral such larvae of claim 1, wherein the collecting the fractions showing transformation activity against coral such larvae.

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