JP4332646B2 - Antifreeze protein derived from fish - Google Patents

Description

  The present invention is a fish that inhabits waters in or around Japan. Spirinchus, Mallotus, Pleurogrammus, Sebastes, Clupea, Limanda, Liopsetta, Clidoderma Genus (Cleisthenes), Microstomus, Lepidopsetta, Platichthys, Kareius, Eupsetta, Gadus, Theragra, Theragra Amphites ces), Ascoldia, or Mholanginpo (Pholidapus), and more specifically, myxocephalus stelleri Tilesius, blue deer (Gymnocanthus herzensteini Jordan et Starks), myxocephalus polyacantallas (Myoxocephalus polyacanthoceus) ), Slugs deer (Hemilepidotus jordani Bean), deer (Furcina osimae Jordan et Starks), deer (Hypomesus pretiosus japonicus (Brevoort)), swordfish (Hypomesus olidus (Dallas)), shishamo (tus) Caraft shishamo (Mallotus villosus (Muller)), Kitano Hocke (Pleurogrammus monopterygius (Pallas)), Willow (Sebastes steindachneri Hilgendorf), Herring (Clupea pallasii Valenciennes), Magdalene (Limanda herzensteini Jordan et Sny) , Black flounder (Limanda schrenki Schmidt), black flounder (Limanda aspera (Pallas)), Roga Rei (Liopsetta obscura (Herzenstein)), Sega Rei (Clidoderma asperrimum (Temminck et Schlegel)), Sow Bee (Cleisthenes pinetorum herzensteini (Schmidt)), Baba Rae (Microctomus achne (Jordan et Starks)), Asa Reef Platichthys stellatus (Pallas)), sea bream (Kareius bicoloratus (Basilewsky)), sand flounder (Eopsetta grigorjewi (Herzenstein)), red sea bream (Gadus macrocephalus Tilesius), walleye (Theragra chalcogramma (Pallas)), tes Hypoptychus dybowskii Steindachner), horse mackerel (Trachurus japonicus (Temminck et Schlegel)), barnyard salmon (Brachyopsis rostratus (Tilesius)), horseshoe (Pholis picta (Kner)), gazi (Opisthocentrus ocellatus (Tilesius), Dragoinpo (Ascoldia variegata knipowitschi Soldatov) or Muroranginpo (Pholidapous dybowskii (Steindachner) ) To produce antifreeze protein from the fish species belonging to.

  From the tissue fluid (blood) of fish that have been caught in the Arctic, Antarctic, and North America offshore to date, it binds specifically to the surface of countless small ice crystals formed in the tissue fluid in the pre-freezing state and deforms into a diamond shape. As a result, a protein called an antifreeze protein (abbreviated as AFP) has been found, which has the function of inhibiting freezing and consequently lowering the apparent freezing point of an aqueous solution containing this protein. These fish include cod (Atlantic cod (Gadus morhua Linneaeus)), flounder (Pseudopleuronexus americanus, English: Winter flounder (Pseudopleuronectes americanus (Walbaum))), herring (English name: Atlantic herring (Clupea harengus Linnaeus)), Cucumber smelt (Osmerus mordax dentex Steindachner), Kajika (Myoxocephalus scorpius, English name: Shorthorn Sculpin (Myoxocephalus scorpius) (Macrozoarces americanus (Block et Schneider)), Myokokephalus octodecenspinos, English name: Longhorn Sculpin (Myoxocephalus octodecemspinosus (Mitchill)), Hemitripterus americanus, English name: Athlantic sea raven (Hemitripterus americanus (Gmelin)) (Fletcher, GL, Hew, CL, and Davies, PL Annu. Rev. Physiol. (2001) 63, 359-390.). As mentioned above, antifreeze proteins are only found in a limited number of fish species, and most of the above fish species are not available in Japan. It is hard to say that the existence of antifreeze proteins has been fully explored for various fish species.

On the other hand, it is known that antifreeze protein is present in body fluids of winter vegetables such as carrots, radish and cabbage, and beetle and moth larvae in addition to fish. Antifreeze proteins extracted from plants have a significantly weaker ability to bind ice crystals than fish-derived proteins. Since antifreeze protein has a function to prevent recrystallization of ice, it is considered that the quality is maintained by mixing it in ice cream, for example. In order to prevent recrystallization more efficiently, it is necessary to mix an antifreeze protein having a stronger ice crystal binding ability, but an effect cannot be expected from a plant having a weak ability. On the other hand, a sufficient effect can be expected for antifreeze proteins derived from fish. However, most of the fish species in which antifreeze proteins have been found so far live in the polar latitudes or in the cold zone, and are caught in large quantities in the temperate zone near Japan, or in large quantities at food supermarkets. It has been desired to separate and purify antifreeze proteins from fish species sold and disposed of and use these antifreeze proteins.
In addition, when using a purified product of fish-derived antifreeze protein in frozen foods, it is necessary to prevent fish-specific odors from adhering to the purified product. Little research has been done on.

   Therefore, an object of the present invention is to find new antifreeze proteins from fish species that are caught in large quantities in the sea near Japan, etc., or sold and discarded in large quantities at food supermarkets. Is to produce a large amount of fish and to prevent the attachment of fishy odor to promote its use.

  In such a situation, the present inventor has conducted intensive research on the body fluid components of fish species caught in or around Japan. Even so, we found for the first time that some of the fish caught especially in winter have antifreeze protein. The present inventor is a fish species that has not been reported to have antifreeze protein so far, Giska deer, Tsukaka deer, Togeka deer, Nameokasika deer, Kinuka deer, Chica, Ishikari Wakasagi, Shishamo, Kitano Hokke, Yanaginomai, Herring, Magdalene, Sunagare, Black-faced Flathead, Scarlet, Black-headed Leopard, Saweye, Bumblebee, Ashbass, Gray Spider, Rockeye, Mussille, Madara, Walleye, Squirrel, Squirrel, Spotted Flyfish, Giganpa Seed tissue fluids and muscle ground fluids, and walleye pollock, komai, shishamo, kalaft shishamo, herring, eelfish, flounder, sold as dry products at food supermarkets, etc. By grinding fish meat dry products such Rumeiwashi Some of the liquid the addition of water, make sure you have components for generating ice crystals of diamond, which has led to the completion of the present invention. Among them, the smelt is a freshwater fish, and there have been no reports of an antifreeze protein that produces diamond-type ice crystals found in freshwater fish. Furthermore, with regard to the attachment of fish odor, which is a problem of antifreeze proteins derived from fish, a new purification method has been established to solve the problem.

That is, the present invention is as follows.
(1) Bluefin deer genus (Gymnocanthus), Yokosuji deer genus (Hemilepidotus), Sarasaka deer genus (Furcina), Smelt genus (Hypomesus), Shiramo genus (Spirinchus), Karafushisukamo (Mallotus), Hokeke genus (Pleurogrammus), Pleurogrammus Sebastes), Herring genus (Clupea), Papilio genus (Limanda), Black flounder genus (Liopsetta), Samegalei genus (Clidoderma), Scorpion genus (Cleisthenes), Buffalo genus (Microstomus), Lepidopsetta genus (Platichthys) ), Kareius, Eopsetta, Theragra, Ammodytes, Hypoptychus, Trachurus, Brachyopsis, Holisis, Pis From fish meat or dry fish of fish species belonging to the genus Opisthocentrus, Zoarces, Ascoldia, or Pholidapus, A method for producing a protein having a function of inhibiting freezing, comprising collecting a protein having a function of inhibiting freezing.
(2) The source of protein is Myss deer (Myoxocephalus stelleri Tilesius), Tsukasaka deer (Gymnocanthus herzensteini Jordan et Starks), Scarlet deer (Myoxocephalus polyacanthocephalus (Pallas)), Namemyo deer (Hemilepidotus jordanios) et Starks), Chica (Hypomesus pretiosus japonicus (Brevoort)), Ishikari Smelt (Hypomesus olidus (Dallas)), Shishamo (Spirinchus lanceolatus (Hikita)), Karafushishamo (Mallotus villosus (Muller)), Kitanourmus pt ), Willow (Sebastes steindachneri Hilgendorf), herring (Clupea pallasii Valenciennes), flounder (Limanda herzensteini Jordan et Snyder), black flounder (Limanda punctatissima (Steindachner)), black flounder (Limanda schrenki ass), Black flounder (Liopsetta obscura (Herzenstein)), Sega rey (Clidoderma asperrimum (Temmi nck et Schlegel)), Sowbee (Cleisthenes pinetorum herzensteini (Schmidt)), Bumblebee (Microctomus achne (Jordan et Starks)), Asahi flounder (Lepidopsetta mochigarei Snyder), Numa flounder (Platichthys stellatus (Pallassky) ), Mussel flounder (Eopsetta grigorjewi (Herzenstein)), Spotted moth (Gadus macrocephalus Tilesius), Walleye (Theragra chalcogramma (Pallas)), Squid (Ammodytespersonatus Girard), Hypoptychus dybowskii Steindus (Tyck) , Whitefish (Brachyopsis rostratus (Tilesius)), western ginkgo (Pholis picta (Kner)), gazi (Opisthocentrus ocellatus (Tilesius)), nagaji (Zoarces elongatus Kindowi) from fish species of fish meat or fish dry matter belonging to a)), it has the ability to inhibit the frozen Characterized in that the collecting protein that, process for producing a protein having the function of inhibiting the freezing.

As described above, antifreeze protein prevents taste deterioration and tissue destruction due to ice crystal growth in, for example, ice cream and frozen foods. In addition, ice freezing protein is used in a cold heat supply system or cold heat storage using ice slurry. It is expected as an effective additive that can eliminate the blockage of the piping system due to crystals. Furthermore, it is a promising substance for cryopreservation and cryopreservation of eggs, sperm and transplanted organs.
However, at present, antifreeze proteins with high potency are found only in specific fish species inhabiting polar regions, so they cannot be produced in large quantities and their effective use has been hindered. On the other hand, the new antifreeze protein of the present invention can be produced from fish species inhabiting Japan, seawater near Japan, and waters of the same climate as the water, and thus can be easily obtained. It greatly contributes to the promotion of the use and development of applied research on antifreeze proteins.

Hereinafter, the present invention will be described in more detail.
The body fluids of fish inhabiting local and deep seas such as the North Pole, South Pole, and their nearby waters do not freeze even when the temperature drops to about minus 2 ° C. In contrast, normal fish body fluids freeze at minus 0.8 ° C. Seawater freezes at minus 1.9 ° C.
It has been clarified that the antifreeze of the body fluids of fishes living in the Arctic, Antarctic, etc. is due to the antifreeze protein or antifreeze glycoprotein (AFGP) produced by these fish themselves. There are four types of antifreeze proteins: AFPI with a molecular weight of about 3,000-4,500, AFPII with a molecular weight of about 20,000, AFPIII with a molecular weight of about 7,000, and a molecular weight of about 11, It is classified into 000 AFPIV. The molecular weight of AFGP is between 2,200 and 33,000. Whether or not the purified protein is AFGP can be easily confirmed by using a Schiff reagent or the like. Each antifreeze protein has a difference in the amino acid composition of the protein and its higher-order structure, and even if it is classified into the same type, the amino acid sequence and higher-order structure of the protein differ depending on the fish species. .
On the other hand, regarding the function of antifreeze protein, normally, when ice nuclei appear in an aqueous solution, ice crystals first grow into a flat hexagonal plate shape. The growth in the direction perpendicular to the plate-like plane is about 100 times slower than the growth in the plate-like plane direction. On the other hand, if antifreeze protein is present in the aqueous solution, the growth of ice crystals in the plane direction of the disk is prevented, and the plate-like body that is formed first is used as the basal plane, perpendicular to the basal plane, Sequentially, smaller plates are stacked and eventually grow slowly into bipyramidal ice crystals with two pyramids.

  Therefore, only when antifreeze protein is present in an aqueous solution collected from a fish species specimen of interest, when the specimen liquid is set to 0 ° C. or lower, the specimen liquid contains a bio-type as shown in FIGS. 1A and 1B. Pyramidal ice crystals, crystallographically, ice crystals called hexagonal bipyramids (FIG. 1A) and polarized tetrahedrons (FIG. 1B) are observed under a microscope. Such bipyramidal ice crystals are formed as a result of the ability of antifreeze proteins to specifically bind to the 12 ice layer planes on the ice crystals. Microscopically, innumerable bipyramidal ice crystals in which the antifreeze proteins in the sample liquid are not connected to each other are generated in a freezing temperature range of 0 ° C. or lower. Macroscopically, this is observed as a non-freezing phenomenon (antifreezing activity) of the specimen. This phenomenon can also be quantified as a freezing point depression or temperature hysteresis of the specimen liquid by using an osmometer (osmometer). In order to find out the presence of antifreeze protein using the freezing point depression measurement method and evaluate its activity, it is necessary to obtain a purified antifreeze protein aqueous solution with high purity. On the other hand, the antifreeze activity evaluation method by observation of bipyramidal ice crystals is observed even if antifreeze proteins exist, even if a large amount of impurities, contaminating proteins, ions, etc. are mixed in the sample liquid. . In addition, if the concentration of antifreeze protein is 0.1 mM or more, bipyramidal ice crystals are observed regardless of the concentration. Therefore, the simplest and quickest method for evaluating whether the specimen, that is, the fish species of interest has antifreeze protein, is to observe bipyramidal ice crystals in the specimen liquid.

In the fish species used as the source for collecting the antifreeze protein of the present invention, the presence of bipyramidal ice crystals in the blood, surimi, or dry sample liquid has been confirmed at all freezing temperatures.
In the present invention, the discovery of antifreeze protein from various fish species inhabiting waters in and around Japan is an epoch-making thing. In view of this result, at least the above, regardless of whether in the northern hemisphere or the southern hemisphere There is a very high probability that there is a fish species that has the ability to synthesize antifreeze proteins in the living body in the water area having a climate equivalent to the water area. The fishing season of the fish species of the present invention is preferably from the end of October to the beginning of May, and particularly preferably from the winter to the beginning of April. In order to obtain antifreeze proteins from these fish species, for example, ion exchange chromatography, gel filtration chromatography, reverse phase chromatography, etc., from the blood of the fish species of the present invention, fish surimi and aqueous extracts of dried products These normal protein separation and purification means can be combined appropriately.

In general, it is considered that proteins in fish bodies are degraded by enzymes and the like immediately after the death of the fish bodies and lose their ability in many cases. Therefore, antifreeze proteins are sold at food supermarkets. It is surprising that it can be purified from fish over time. In the present invention, the fact that antifreeze proteins can be sufficiently purified from fish sold at food supermarkets, etc., and further, even from products of dried fish that have undergone treatment such as heating, washing, drying, etc. Finding that frozen protein can be purified is also very innovative. These facts are based on the very high stability of fish-derived antifreeze proteins, and because of this rare nature, antifreeze proteins are purified from suspensions of fish surimi or fish grounds. In this step, high-temperature heat treatment can be applied.
The unpleasant odor of fish is caused by the decay of the organs and their contents in the head and abdomen over time and the smell inherent in the fish meat itself. Therefore, it is basically desirable to remove the head and abdomen at an early stage in a fish body that is a raw material for antifreeze protein purification. The fish body from which the abdomen and head are removed can be dried and stored in addition to refrigerated storage. This drying has the effect of concentrating the antifreeze protein and the effect of suppressing the growth of bacteria, and is beneficial in terms of reducing the weight of the fish during storage. Moreover, since drying enables storage at room temperature, it is advantageous in terms of storage, transportation, export to overseas, and the like.

In the present invention, the antifreeze protein may be obtained by applying the above-described commonly used protein separation and purification method. However, in order to obtain a suitable antifreeze protein as a food additive without fishy odor, 1 2) A step of preparing a fish paste or a dried fish suspension 2) A step of centrifuging a fish paste or a dried fish suspension to obtain a supernatant 3) A step of heat-treating the supernatant 4) A step of removing the precipitate produced by 3) by centrifugation to obtain a supernatant containing antifreeze protein, 5) and a step of recovering antifreeze protein from the supernatant obtained in 4 in order, It is effective to use an antifreeze protein purification method.
That is, in the process of 1), the fish meat is ground, or the dried fish is finely cut with scissors and then pulverized with a mixer, and water or an aqueous solution of ammonium bicarbonate or sodium hydrogen phosphate is added thereto. A fish suspension is used. Thereby, antifreeze protein is eluted in an aqueous liquid. The fish meat surimi may be obtained by chopping the fish meat and using a mixer, but may be obtained by crushing with a surimi production machine by a conventional method. In the step 2), the surimi suspension or the suspension of the dried fish pulverized product is centrifuged to obtain a supernatant containing antifreeze protein. Centrifugation conditions are 3,000 to 12,000 revolutions / minute and 5 to 60 minutes.

  Thereafter, as the step 3), the supernatant obtained in the above step is subjected to heat treatment. This heat treatment effectively removes or reduces the odor peculiar to fish and surimi and It is a protein that denatures and precipitates coexisting proteins other than frozen proteins. For this reason, it can be said that the higher the temperature set in the heat treatment, the higher the temperature in the temperature range of 100 ° C. or lower, as long as the target antifreeze protein is not denatured and precipitated. Anti-freeze proteins of Gizika deer, Spruce deer, Bluefin deer, Name yokos deer deer, Kinuka deer are particularly resistant to heat, so that these surimi suspensions are free from odor by heating at 90 ° C for 10 minutes. An antifreeze protein component could be obtained. When using these fish species, heat treatment for 10 minutes to 30 minutes in a temperature range of 70 ° C. to 98 ° C. is preferable.

On the other hand, Chika, Ishikari Wakasagi, Shishamo, Kitano Hokke, Willow, Myring, Magdalene, Sungarle, Black-faced Flathead, Scarlet, Black-headed Gray, Samegalei, Sowbee, Babaray, Asagarei, Numagarei, Ishigora, Sandaikoi , Gazi, Nagagaji, Droginpo, and Muranginpo were subjected to a heat treatment process in the range of 50 to 70 degrees C for 10 to 30 minutes. Even at this temperature, the fish odor was removed and the purity was over 60%. An antifreeze protein preparation was obtained. The purified antifreeze protein preparation can be said to have sufficient quality depending on the application.
In the step 4), the heat-treated supernatant is centrifuged to remove precipitated contaminating proteins. The obtained supernatant containing the antifreeze protein at a high concentration may be used in the form as it is, but is preferably used as a dry powder by freeze-drying.

As described above, the antifreeze protein of the present invention has a function of preventing recrystallization of ice, so that the antifreeze protein obtained from each of the above fish species can be any type of AFPI to IV. Regardless of whether it is classified as frozen protein or not, it can be used as an anti-recrystallization inhibitor or freezing inhibitor of ice, and can be used to maintain its quality by mixing into ice cream or frozen food, for example. This quality maintenance effect can also be expected when meat, vegetables, cells (egg or sperm), tissues, organs, etc. are cryopreserved for a long time. Furthermore, in recent years, a cold energy supply system or a cold energy storage system using ice slurry having a large energy density as a heat medium has been proposed. However, in these, there is a problem of blockage of the piping system due to ice recrystallization, and the present invention. This antifreeze protein effectively prevents recrystallization of ice and can be a promising means to solve this problem. In addition, by incorporating a gene encoding an antifreeze protein into a plant body, it can be expected as an applied technology that the plant has cold resistance.

Examples of the present invention will be described below, but the present invention is not particularly limited thereby.

Example 1
<Confirmation of antifreeze protein>
(1) Specimen sample The fish species used as the source of antifreeze protein are as follows.

Gisukajika (Myoxocephalus stelleri Tilesius, English name Frog Sculpin):
Catch on the coast of Rishiri Island.
Black deer (Gymnocanthus herzensteini Jordan et Starks, English name Black edged sculpin): caught on the coast of Rishiri Island.
Togekajika (Myoxocephalus polyacanthocephalus (Pallas), English name Great Sculpin): Sapporo, Hokkaido Atsubetsu District Atsubetsunishi Article 4 2-chome 752 address 318 Cowboy (CowBoy) Atsubetsu I FujiTomi fisheries, and purchased from Hokkaido Notsuke Fisheries Cooperative Association.
Chica (Hypomesus pretiosus japonicus (Brevoort)):
Purchased from Akibetsu I Fujitomi Fisheries Co., Ltd., Hokkaido Boys and Fisheries Cooperatives, 318 Cowboy (CowBoy) 752-52 Atsubetsu Nishi, Atsubetsu-ku, Sapporo, Hokkaido.
Nagagaji (Zoarces elongatus Kner, fame Notched-fin eelpout):
Purchased from Hokkaido Notsuke Fishery Cooperative.
Pond smelt (Hypomesus olidus (Dallas), English name Freshwater smelt):
Purchased from Ibaradogawa Smelt Fishing Spot in Kita-ku, Sapporo.

Yellow striped flounder (Limanda herzensteini Jordan et Snyder, English name Brown Sole):
Purchased from Cowboy (CowBoy) Atsubetsu 1 Fujitomi Suisan, Awabetsu West 4-2-75-2, Atsubetsu-ku, Sapporo, Hokkaido.
Sunagarei (Limanda punctatissima (Steindachner), English name Longsnout flounder):
Purchased from Cowboy (CowBoy) Atsubetsu 1 Fujitomi Suisan, Awabetsu West 4-2-75-2, Atsubetsu-ku, Sapporo, Hokkaido.
Kurogarei (Liopsetta obscura (Herzenstein), English name Black plaice):
Purchased from Cowboy (CowBoy) Atsubetsu 1 Fujitomi Suisan, Awabetsu West 4-2-75-2, Atsubetsu-ku, Sapporo, Hokkaido.
Samegarei (Clidodema asperrimum (Temminck et Schlegel) , English name Roughscale sole):
Purchased from Cowboy (CowBoy) Atsubetsu 1 Fujitomi Suisan, Awabetsu West 4-2-75-2, Atsubetsu-ku, Sapporo, Hokkaido.
Sawbee (Cleisthenes pinetorum herzensteini (Schmidt), English name Pointhead flounder):
Purchased from Cowboy (CowBoy) Atsubetsu 1 Fujitomi Suisan, Awabetsu West 4-2-75-2, Atsubetsu-ku, Sapporo, Hokkaido.

Babagarei (Microstomus achne (Jordan et Starks), English name Slime flounder):
Purchased from Cowboy (CowBoy) Atsubetsu 1 Fujitomi Suisan, Awabetsu West 4-2-75-2, Atsubetsu-ku, Sapporo, Hokkaido.
Aspidfish ( Lepidopsetta mochigarei Snyder)
Purchased from Cowboy (CowBoy) Atsubetsu 1 Fujitomi Suisan, Awabetsu West 4-2-75-2, Atsubetsu-ku, Sapporo, Hokkaido.
Mushigarei (Eopsetta grigorjewi (Herzenstein), English name Shothole halibut):
Catch on the coast of Ishikari Bay, Hokkaido.
Madara (Gadus macrocephalus Tilesius, English name Pacific cod):
Purchased from Cowboy (CowBoy) Atsubetsu 1 Fujitomi Suisan, Awabetsu West 4-2-75-2, Atsubetsu-ku, Sapporo, Hokkaido.
Name sword pin (Hemilepidotus jordani Bean, English name Yellow Sculpin):
Catch on the coast of Samani-cho, Hokkaido.
Shiwaikanago (Hypoptychus dybowskii Steindachner, English name Naked sand lance):
Catch on the coast of Rishiri Island, Hokkaido.
Dragoinpo (Ascoldia variegata knipowitschi Soldatov):
Catch at the coast of Samani-cho, Hokkaido

Alaska pollack (Theragra chalcogramma (Pallas), English name Walleye Pollock):
Purchased from Cowboy (CowBoy) Atsubetsu 1 Fujitomi Suisan, Awabetsu West 4-2-75-2, Atsubetsu-ku, Sapporo, Hokkaido.
Kitano Hocke (Pleurogrammus monopterygius (Pallas), English name Atka mackerel):
Catch on the coast of Ishikari Bay, Hokkaido.
Willow (Sebastes steindachneri Hilgendorf):
Catch on the coast of Ishikari Bay, Hokkaido.
Herring (Clupea pallasii Valenciennes, California herring):
Purchased from Cowboy (CowBoy) Atsubetsu 1 Fujitomi Suisan, Awabetsu West 4-2-75-2, Atsubetsu-ku, Sapporo, Hokkaido.
Shishamo (Spirinchus lanceolatus (Hikita), English name Sishamo Smelt):
Purchased from Akibetsu 1 Fujitomi Suisan, 318 Cowboy (CowBoy), Atsubetsu West 4-2-75-2, Atsube-ku, Sapporo, Hokkaido, and caught on the coast of Yodogawa Town, Hokkaido.
Limanda schrenki Schmidt (English name: Cresthead flounder):
Catch on the coast of Ishikari Bay, Hokkaido.
Flounder (Platichthys stellatus (Pallas), English name Starry flounder):
Catch on the coast of Ishikari Bay, Hokkaido.

Lily aspera (Pallas), English fin sole):
Catch on the coast of Ishikari Bay, Hokkaido.
Ishigarei (Kareius bicoloratus (Basilewsky), English name Stone flounder):
Purchased from 148 Cowboy (CowBoy) Atsubetsu 1 Fujitomi Suisan, Atsube-ku, Atsubetsu-ku, Sapporo, Hokkaido, and caught on the coast of Ishikari Bay, Hokkaido.
Kinka deer (Furcina osimae Jordan et Starks, English name Silk Sculpin):
Catch on the coast of Tomakomai, Hokkaido.
Sand lance (Ammodytes personatus Girard, English name Japanese sand lance):
Catch on the coast of Ishikari Bay, Hokkaido.
Muroranginpo (Pholidapous dybowskii (Steindachner), English name Dybowsky's gunnel): caught on the coast of Ishikari Bay, Hokkaido.
Whitefish (Brachyopsis rostratus (Tilesius), English Longsnout poacher): caught in Notsuke Bay, Hokkaido.
Nishiki Gimpo (Pholis picta (Kner), English Painted gunnel): caught in Notsuke Bay, Hokkaido.
Gazi (Opisthocentrus ocellatus (Tilesius), English name Redspotted gunnel): caught in Notsuke Bay, Hokkaido.
Caraft Shishamo (Mallotus villosus (Muller), English name Atlantic capelin): Purchased from Cowboy (CowBoy) Atsubetsu 1 Fujitomi Suisan, 3F 752, Atsubetsu Nishi, Atsubetsu-ku, Sapporo, Hokkaido.
Jack mackerel (Trachurus japonicus (Temminck et Schlegel, English name Yellowfin horse mackerel)): Sapporo, Hokkaido Atsubetsu District Atsubetsunishi Article 4 2-chome 752 address 318 Cowboy (CowBoy) Atsubetsu 1 FujiTomi purchased from fisheries.

The dried fish products used as the source of antifreeze protein are as follows.
Walleye (brand name: Musiritara, manufacturer: Daito Foods) Purchased from Cowboy Atsubetsu 1 at 352, 2-75-2 Atsubetsu Nishi, Atsubetsu-ku, Sapporo, Hokkaido.
Shishamo (Brand name: Domestic Hime Shishamo, Manufacturer: Daimaru Suisan) Purchased from Cowboy Atsubetsu 1 at 352-2 Amebetsu Nishi, Article 2, 2-chome, Atsubetsu-ku, Sapporo, Hokkaido.
Ikanago (Brand name: Onago smoked, manufacturer: Fujimizu) Purchased from Cowboy Atsubetsu 1 at 352, 2-75-2 Atsubetsu Nishi, Atsubetsu-ku, Sapporo, Hokkaido.
Koganegaray (trade name: Roll Kogane Kai, manufacturer: Daito Foods) Purchased from Cowboy Atsubetsu 1 at 352, 2-75-2 Atsubetsu Nishi, Atsubetsu-ku, Sapporo, Hokkaido.
Urumei eagle (trade name: Urume Niboshi, manufacturer: Kane Nana) Purchased from Cowboy Atsubetsu 1 at 352, 2-75-2 Atsubetsu Nishi, Atsubetsu-ku, Sapporo, Hokkaido.

(2) Observation of bipyramidal ice crystals a) For fresh fish of the above-mentioned fish species, while keeping the gills open, the head and head are held down to prevent damage to the liver, small intestine and other organs. Blood was collected by carefully inserting a syringe needle attached to a 5 ml syringe into the triangular red-and-black heart part visible on the side. The total amount of blood collected was 150 ml for each species of Giska deer, Tsukadeka deer and Chica, 420 ml for Stagka deer, and 30 ml for Kitano Hokke and Herring. Also, 20ml each for flounder, sunbed bream, black flounder, stag beetle, bubbock bream, black flounder, black flounder, blackhead bream, flounder, prickly flounder, 35ml for madara and walleye, 25ml for sludge, stag beetle, red flounder, flounder It was. These were used in the following tests (b) and Example 2. For fresh fish other than the above, Ishikari Wakasagi, Yanaginomai, Sawachi, Shishamo, Caraft Shishamo, Shiwai Kanago, Ikanago, Shichirouuo, Nishiki Gimpo, Gazi The crushed material was used as a surimi. An equal amount (20 ml) of 0.1 M ammonium sulfate aqueous solution (ph7.9) was added to 20 ml of surimi and suspended. Further, dry fish products of walleye pollock, shishamo, squid, flounder, urchin eagle, and horse mackerel are chopped to about 1 cm with scissors, pulverized with a mixer, 20 ml of 0.1 M ammonium sulfate aqueous solution (pH 7 .9) was added and mixed well, and the mixture was allowed to stand at 4 ° C. overnight to obtain a dry matter suspension. The blood, surimi suspension, and dry matter suspension thus obtained were used in the following tests (b) and Example 2.

It should be noted that the evaluation of whether or not the sample liquid of interest contains antifreeze protein can be performed by conducting bipyramidal ice crystal observation experiments under a low-temperature microscope even with only 1 ul of liquid.
b) 1 ul of each blood sample collected as described above was dropped onto a cover glass having a diameter of 16 mm of a Leica DMLB100 microscope (Leica DMLB 100 photomicroscope). This was directly sandwiched by another cover glass having a diameter of 12.5 mm and set in a cooling box installed on the stage of a DMLB100 microscope. A light intake hole with a diameter of 1 mm was formed on the top and bottom of the cooling box, and light from the microscope light source passed through the box from the lower hole and entered the lens through the upper hole. By setting the sample liquid on the optical axis defined by the upper and lower holes, the substance in the sample liquid on the optical axis can be observed with a microscope. The temperature in the cooling box in which the sample liquid is set is controlled with an error of +/− 0.1 ° C. by an LK600 temperature controller (LK600 temperature controller) manufactured by Linkham. After the sample liquid was set at room temperature, the temperature in the cooling box was lowered to 0.2 degrees C per second to minus 22 degrees C by the temperature controller. The entire specimen liquid freezes at some temperature between about minus 1.4 degrees C and minus 2.2 degrees C. After freezing, the temperature in the cooling box is raised at 0.1 ° C. per second, and the rise is stopped at 0 ° C. If the temperature is kept at 0 ° C. for about 1 to 10 seconds, the freezing melts and countless cracks After passing through two ice crystal states, it was observed that a certain amount of ice crystal crush floated in the water. At that moment, the temperature in the cooling box was lowered to about minus 0.1 degree C minus minus 1.0 degree C and stopped, and the shape of ice crystals was observed. The observation results are shown in FIGS. Bipyramidal ice crystals were observed in all of the fish species used in the test, and it was confirmed that all of these fish species had antifreeze proteins. In addition, similar tests were performed using 1 μl of fish meat surimi suspension and 1 μl of dry matter suspension. In these tests, bipyramidal ice crystals were observed for all fish species.

Example 2
<Separation and purification of antifreeze protein 1>
a) Purification of antifreeze protein In the same manner as in Example 1 (2) a), 30 ml of blood was collected from Gizika deer into a plastic test tube. 30 ml of Gizzard deer blood collected in a plastic test tube was left in a refrigerated chamber at 4 degrees C overnight to clot and precipitate only blood cell components (generally referred to as clotting operation). Approximately 30 ml of blood (15 ml) was precipitated as blood cells, and only 15 ml of supernatant serum was collected. At this time, centrifugation was performed at 3000 rpm for 15 minutes using a concentrating centrifuge to improve the separation ability of blood cells and serum. At this time, bipyramidal ice crystals were observed in 15 ml of the collected serum to confirm the presence of antifreeze protein. The 15 ml of serum was subjected to gel filtration by Sephadex G-50 gel column chromatography with an inner diameter of 2.5 cm and a height of 96 cm (volume: 475 ml). A 0.1 M aqueous ammonium sulfate solution (pH = 7.9) was used as an eluent. FIG. 9 shows a 280 nm ultraviolet absorption pattern for the serum gel filtration fraction. 10 ml of gel filtration fraction was obtained per test tube. They were then lyophilized.

  After adding 0.5 to 2 ml of 0.1M ammonium sulfate aqueous solution (pH = 7.9) to the freeze-dried product of each fraction, bipyramidal ice crystals are observed for each, and antifreeze protein is contained. The fractions from No. 10 to No. 24 (shaded part of FIG. 9: Antifreeze Active Fractions) were used for the next purification step. A solution of fractions from No. 10 to No. 24 dissolved in about 45 ml of ammonium sulfate aqueous solution (pH = 7.9) is used for an aqueous solution of protein having a molecular weight of 30,000 or less containing antifreeze protein by using Millipore Amicon Centriprep. 40 ml was obtained. Add 5 ml of Pharmacia DEAE Sepharose CL-6B anion exchange resin equilibrated with 0.1 M ammonium sulfate aqueous solution (pH = 7.9), and centrifuge for 1 minute at 3000 rpm in a concentrating centrifuge. About 40 ml of an antifreeze protein aqueous solution having non-binding properties was obtained. To this aqueous solution, 5 ml of Pharmacia CM Sepharose CL-6B cation exchange resin equilibrated with 0.1 M ammonium sulfate aqueous solution (pH = 7.9) was added, and this cation exchange was performed by centrifuging at 3000 rpm for 1 minute. An antifreeze protein aqueous solution having non-binding property to the resin was also obtained.

This sample was lyophilized, dissolved in a small amount of 0.1 M aqueous ammonium sulfate (pH = 7.9), and fractionated by reverse phase HPLC chromatography using an ODS (C8) column. Equilibration between the HPLC route and the column was performed using a 0.1% aqueous trifluoroacetic acid solution (liquid A), and elution was performed using acetonitrile (liquid B) containing 0.1% trifluoroacetic acid. The concentration gradient of solution B was set to a linear increase of 1% per minute, and the solution feeding speed was set to 1 ml / min. FIG. 10 shows this reverse phase HPLC pattern. Each peak observed in FIG. 10 was collected and freeze-dried, and then each was dissolved in a small amount of 0.1M ammonium sulfate aqueous solution (pH = 7.9) to examine whether bipyramidal ice crystals were formed. As a result, it has been found that AFP1 produces a dihedral tetrahedral ice crystal shown in FIG. 1B, while AFP2 produces a hexagonal bipyramidal ice crystal shown in FIG. 1A. Thus, lyophilized products of two peak fractions marked with AFP1 and AFP2 were obtained as purified antifreeze protein powder.
It was confirmed by electrophoresis shown below that these two are different antifreeze proteins.

b) Gel electrophoresis SDS gel electrophoresis was performed on AFP1 and AFP2 derived from Giska deer. Since the molecular weights of AFP1 and AFP are expected to be about several thousand to 50,000, 16% was selected as the SDS concentration in the gel in order to migrate proteins with good separation. The composition of the reagent used for electrophoresis is as follows.
Separation gel (10 ml)
2.0 ml water
5.4 ml 30% polyacrylamide + 0.8% bisacrylamide
2.5 ml 1.5M Tris-HCl (pH = 8.8)
120 μl 10% sodium dodecyl sulfate solution
70 μl 10% ammonium persulfate solution
15.0 μl N, N, N ', N'-tetramethylenediamine solution
Concentrated gel (5 ml)
3.17 ml water
500 μl 30% polyacrylamide + 0.8% bisacrylamide 30%
1.25 ml 0.5M Tris-HCl (pH = 6.8)
50 μl 10% sodium dodecyl sulfate solution
30 μl 10% ammonium persulfate solution
7.5 μl N, N, N ', N'-tetramethylenediamine solution

  As molecular weight marker (Fig. 11, Marker1), GIBCO protein molecular weight standard for low molecular weight was used, and dimeric antifreeze protein RD3 (molecular weight 14kDa, Marker2), monomeric for comparison with similar proteins Antifreeze protein RD3Nl (6.5 kDa, Marker3), chicken egg white lysozyme (molecular weight 14.3 kDa, Marker4), and antifreeze glycoprotein (3 kDa, Marker4) were used. All proteins other than the marker are dissolved in 7ul of 0.1M ammonium sulfate aqueous solution (pH = 7.9), and 7ul of buffer solution (0.065M Tris-HCl (pH = 6.8), 2% SDS, 10% Sucrose, 5% β -mercapthoethanol, 0.001% BPB staining reagent) and then boiled at 95 ° C for 5 minutes to obtain an electrophoresis sample. This was subjected to electrophoresis for 2 hours under a constant voltage of 100 V using a mini slab gel electrophoresis apparatus manufactured by Nippon Aido. The result is shown in FIG. As shown in the figure, AFP1 was an antifreeze protein having a molecular weight of about 11 kDa and AFP2 having a molecular weight of 3-6 kDa. From the viewpoint of these molecular weights, it is estimated that AFP1 belongs to the above-mentioned type IV (AFPIV) and AFP2 belongs to type I (AFPI).

Example 3
<Separation and purification of antifreeze protein 2>
According to the procedure described in Example 1 (2), 40 ml of Chica Surimi suspension was prepared. This was put into a plastic test tube and centrifuged at 10,000 rpm for 15 minutes to obtain about 20 ml of supernatant. The protein concentration of this supernatant was about 25 mg / ml. The plastic test tube containing the supernatant was immersed in a 70 ° C. bath for 10 minutes for heat treatment. After removing the plastic test tube from the warm bath, it was immediately put into crushed ice and quenched. Thereafter, the sample was centrifuged at 18,000 rpm for 30 minutes to obtain about 15 ml of supernatant. The protein concentration of this supernatant was about 6.6 mg / ml. FIG. 12 shows the result of SDS gel electrophoresis (FIG. 12A) for this supernatant and the ultraviolet absorption pattern (FIG. 12B) of Glu column chromatography. SDS gel electrophoresis was performed under the same conditions as in FIG. 9, and glu column chromatography was performed by eluting 200 μl of the sample solution at 0.2 ml / min with an Amersham Pharmacia Biotech Sepharose 6 HR10 / 30 column. AFP3 (molecular weight of about 17 kDa), AFP4, and AFP5 (molecular weight of about 14 kDa) shown in the rightmost lane of the electrophoretic diagram of FIG. 12A are all antifreeze proteins derived from Chica that generate bipyramidal ice crystals having the shape of FIG. 1A. there were. Judging from these molecular weights, it is estimated that all Chica-derived antifreeze proteins belong to type II. The fractions of gel chromatography corresponding to these AFP3, AFP4, and AFP5 are the portions indicated by the range of arrows in FIG. 12B. By comparing the absorption peak intensity of this part with that of the rest, it is possible to purify the chica-derived antifreeze protein with a degree of purification of about 62% by obtaining a centrifugal supernatant after heat treatment at 70 ° C. for 10 minutes. It has been shown.

  As for the fish surimi suspension obtained from Gizika deer, the antifreeze protein was separated and purified including gelation and gel electrophoresis in the same manner as described above. In this case, however, the antifreeze protein that seems to be mainly type I was obtained. It was. This antifreeze protein could be obtained by heating at 90 ° C.

Example 4
{Separation and purification of antifreeze protein 3}
According to the procedure described in Example 1, (2), 40 ml of a dried shishamo suspension was taken into a plastic test tube. Add 0.4 ml of 99.5% acetic acid to this, mix well, and leave at 4 ° C for 1 hour. The acidic precipitate produced by this operation was removed by centrifuging at 18,000 rpm for 30 minutes to obtain about 35 ml of supernatant. To this, 0.5 ml of 1M sodium hydroxide aqueous solution was added and mixed well, and the mixture was left at 4 ° C. for 1 hour. The basic precipitate produced by this operation was removed by centrifuging at 18,000 rpm for 30 minutes to obtain about 32 ml of supernatant. This solution was subjected to separation and purification of antifreeze protein including a heat treatment step in the same manner as described above to obtain antifreeze protein considered to be type II.

Example 5
{Separation and purification of antifreeze protein 4}
According to the procedure described in Example 1 (2), 40 ml of Nagagaji Surimi suspension was prepared. This was put into a plastic test tube and centrifuged at 6,000 rpm for 30 minutes to obtain about 20 ml of supernatant. This supernatant was dialyzed against 50 mM sodium acetate buffer (pH = 3.7) to aggregate the contaminating proteins. This was removed by centrifugation at 12,000 rpm for 30 minutes to obtain a supernatant. The supernatant was subjected to cation exchange chromatography, and 1 ml of eluate was collected by a fraction collector while detecting absorption at 280 nm. Amersham Pharmacia Biotech FPLC system and BIO-RAD High-S column were used for cation exchange chromatography. The column was equilibrated and the supernatant was taken up using 50 mM sodium acetate buffer (pH = 3.7), and elution was performed by applying a linear gradient of 0 to 0.5 M sodium chloride at a flow rate of 1 ml / min. All operations up to this point were performed at 4 degrees C. Next, the sample solution in which absorption at 280 nm was observed was purified by reverse phase chromatography using a TOSO HPLC system and an ODS column. The column was equilibrated and sample solution was taken up using 0.1% trifluoroacetic acid, and a linear gradient of acetonitrile was used for elution. The absorbance of the eluted sample was detected at 214 nm and 280 nm, and after obtaining an eluate fraction containing a single protein, this was lyophilized. This lyophilized powder was dissolved in 0.1M ammonium bicarbonate aqueous solution, and the bipyramidal ice crystals were observed to confirm that it was an antifreeze protein. This antifreeze protein sample was digested with Aspaginylendopeptidase and Trypsin, and the peptide fragments obtained for each were again collected by reverse phase chromatography. About these peptide fragments, the amino acid sequence was determined by using the amino acid sequence analyzer comprised from Applied Biosystems 491 Protein Sequencer, 785A Programmable Absorbace Detector, and 140C Microgradient System. As a result, Nagagaji expresses three proteins with similar sequences and consisting of 65, 66 and 67 residues.
The amino acid sequences of these three types of antifreeze proteins and the base sequence of the CDS region of the gene are shown in SEQ ID NOs: 1 to 6, respectively. These base sequences showed a homology of about 75 to 90% with a type III antifreeze protein derived from Macrozoalkers americanus whose base sequence has already been clarified. In this way, it was shown that type III antifreeze protein was purified from Nagareji surimi.

The purification method shown in Example 1 is applied to the blood, surimi, or dry matter suspension of the fish species used in the present invention to purify to a single band on SDS electrophoresis. The results of the identification of antifreeze protein types from type I to IV for the following fish species are shown below.
a) Type I Giska deer, Shark deer, Thorn deer, Name yoko deer,
Kinka deer, flounder, sun flounder, black flounder, flounder,
Blackhead flounder, sage lei, saw bee, bubba flounder,
Asaba flounder, flounder, flounder, flounder b) Type II Chica, Ishikari Smelt, Shishamo, Karafushishimo, Herring,
Japanese horse mackerel, white-tailed fish c) Type III type Nagagaji, Doroginpo, Muroranginpo, Nishikiginpo, Gazi d) Type IV type Giska deer, Tsukajika deer, Stagka deer, Nameyo Kosika deer,
Kinuka deer (Note. These express both type I and type IV)
e) AFGP
Madara, walleye

It is a schematic diagram which shows the shape of a bipyramid type ice crystal. It is the microscope picture of the bipyramid type | mold ice crystal observed in the blood of Giska deer, Swordfish, Tsukaka deer, Chica, Ishikari Smelt and Nagagaji. It is the microscope picture of the bipyramid type | mold ice crystal observed in the blood of a flounder, a blackhead flounder, a sun flounder, a red flounder, a shark, and a bee. It is the microscope picture of the bipyramid type | mold ice crystal observed in the blood or surimi suspension of walleye pollack, Kitano Hocke, willow tree, herring, shishamo, and black flounder. It is the microscope picture of the bipyramid type | mold ice crystal observed in the blood or surimi suspension of a marsh flounder, a flounder, Ishigarei, Kinuka deer, Ikanago, and Muroranginpo. It is the microscope picture of the bipyramid type | mold ice crystal observed in the blood or surimi suspension of a codfish, Babagarei, Namekojika deer, Doroginpo, Shiwaikanago, and Musugarei. It is the microscope picture of the bipyramid type | mold ice crystal observed in the surimi suspension of Nishikigimpo, Gazi, Shiro-Uo, and Karafushishishamo. It is the microscope picture of the bipyramid type | mold ice crystal observed in the suspension of the dry product crushed product of Urumei eagle, horse mackerel, walleye pollock, shishamo, and flounder. It is a figure which shows the absorption pattern at the time of irradiating a 280-nm ultraviolet-ray with respect to the gel filtration fraction of Gizika deer serum. It is a figure which shows the elution pattern of the reverse phase HPLC chromatography about the 10th-20th of the said gel filtration fraction. It is a figure which shows the result of the SDS gel electrophoresis about the fraction AFP1 and AFP2 obtained by this reverse phase HPLC chromatography. A. It is a figure which shows the result of the SDS gel electrophoresis about the centrifugation supernatant before and after the heat processing for 10 minutes at 70 degreeC with respect to the surimi suspension of Chica. B. It is a figure which shows the elution pattern of the gel chromatography with respect to the sample solution after heat processing for 10 minutes at 70 degreeC.

Claims (2)

Species (Gymnocanthus), Yokosuji deer (Hemilepidotus), Sarasaka deer (Furcina), Smelt (Hypomesus), Shiramo (Spirinchus), Karafushishamo (Mallotus), Hokke (Pleurogrammus), Sebar, Sebar Herring genus (Clupea), Papilio genus (Limanda), Black flounder genus (Liopsetta), Sega genus genus (Clidoderma), Scorpion genus (Cleisthenes), Buffalo genus (Microstomus), Lepidopsetta, Platichthys, Ishiga rei The genus (Kareius), the genus Euopetta, the theragra, the Ammodytes, the Hypoptychus, the Trachurus, the Brachyopsis, the Phoris, Opisthocentrus), Nagagaji genus (Zoarces), from Doroginpo genus (Ascoldia), or Muroranginpo genus (Pholidapus) belonging to fish species of the fish meat or fish dry matter, the frozen And recovering a protein having the function to harm, process for producing a protein having the function of inhibiting the freezing. The source of the protein is geese deer (Myoxocephalus stelleri Tilesius), stag beetle (Gymnocanthus herzensteini Jordan et Starks), stag (Deer) , Chica (Hypomesus pretiosus japonicus (Brevoort)), Ishikari Smelt (Hypomesus olidus (Dallas)), Shishamo (Spirinchus lanceolatus (Hikita)), Karafushishamo (Mallotus villosus (Muller)), Kitanohokke (Pleurmus mussel) (Sebastes steindachneri Hilgendorf), herring (Clupea pallasii Valenciennes), flounder (Limanda herzensteini Jordan et Snyder), flounder (Limanda punctatissima (Steindachner)), black flounder (Limanda schrenki Schmidt), black flounder (Polia) obscura (Herzenstein)), Samerei (Clidoderma asperrimum (Temminck et Sch legel)), Sowbee (Cleisthenes pinetorum herzensteini (Schmidt)), Babagarai (Microctomus achne (Jordan et Starks)), Asahagaray (Lepidopsetta mochigarei Snyder), Numagarai (Platichthys stellatus (Pallas)), Ishilewi Mussel flounder (Eopsetta grigorjewi (Herzenstein)), spotted moth (Gadus macrocephalus Tilesius), walleye pollock (Theragra chalcogramma (Pallas)), sand eel (Ammodytespersonatus Girach) (Hypoptychus dybowskii Steindachus) (Brachyopsis rostratus) from fish species of fish meat or fish dry matter belonging to, data having a function of inhibiting the frozen And recovering the Park protein, method for producing a protein having the function of inhibiting the freezing.