WO2004104201A1 - Proteine antigel trouvee dans le poisson - Google Patents

Proteine antigel trouvee dans le poisson Download PDF

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Publication number
WO2004104201A1
WO2004104201A1 PCT/JP2004/007105 JP2004007105W WO2004104201A1 WO 2004104201 A1 WO2004104201 A1 WO 2004104201A1 JP 2004007105 W JP2004007105 W JP 2004007105W WO 2004104201 A1 WO2004104201 A1 WO 2004104201A1
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protein
amino acid
acid sequence
seq
antifreeze
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PCT/JP2004/007105
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English (en)
Japanese (ja)
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Yoshiyuki Nishimiya
Ai Miura
Sakae Tsuda
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National Institute Of Advanced Industrial Science And Technology
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Publication of WO2004104201A1 publication Critical patent/WO2004104201A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K5/00—Heat-transfer, heat-exchange or heat-storage materials, e.g. refrigerants; Materials for the production of heat or cold by chemical reactions other than by combustion
    • C09K5/20—Antifreeze additives therefor, e.g. for radiator liquids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish

Definitions

  • the present invention relates to an antifreeze protein obtained by newly preparing from body fluids and surimi of a long-tailed fish (Zoarces elongatus Kner) or a wild fish (Brachyopsi s rostratus (Ti lesius)), which are fish species captured in the waters around Japan.
  • the present invention also relates to an amino acid sequence of the protein, a DNA encoding the protein, a vector containing the DNA, a transformant containing the vector, and the like.
  • ⁇ In the freezing temperature range, only water molecules associate with each other to form ice blocks in accordance with the law of nature inside any substance containing water (hydrate). At this time, all substances other than water molecules are forced to move due to physical pressure caused by the formation of ice blocks, and are driven to a position other than the ice blocks in the hydrated substance (called freeze concentration). Therefore, thawing a hydrated substance that has undergone freeze-concentration impairs the internal structure, physiological activity, or flavor before freezing.
  • Antifreeze protein (AFP) which has an antifreeze function, has the ability to strongly suppress freeze-concentration of hydrates, and exhibits properties such as inhibition of ice recrystallization and thermal hysteresis.
  • AFP has an effect of maintaining the quality of frozen foods and the like and preventing the loss of the physiological activity of cells and organs due to freezing. It is also expected to be used as an effective additive that can eliminate the clogging of the piping system due to ice recrystallization in cold heat supply systems or cold heat storage using ice slurry. In addition, attempts have been made to apply AFP to improving the cryopreservation resistance of meat, vegetables, processed foods, blood, cells, eggs, sperm, transplanted organs, and the like.
  • the present invention is intended to solve such a conventional problem, and it is possible to search for AFP for fish species that can be caught in large quantities in the waters near Japan, etc. It purifies AFP or supplies it stably throughout the year using techniques such as gene expression and chemical synthesis to provide a new protein or peptide with antifreeze ability.
  • Nagagaji or Shichirou-o is caught in large quantities together with the North Sea prawns when fishing using bottom seine, separated, collected into waste fish rigos and discarded by the disposal company.
  • Other fish species, such as smelt with antifreeze protein, are traded at a cost of more than ⁇ 300 to ⁇ 2,000 per kilogram, while Nagagazi or Shichirowo costs $ 0 per kilogram. Therefore, Nagagaji or Shichirou-o is the most suitable fish species as a raw material for recovering and refining antifreeze proteins and keeping them at a low price.
  • the present inventor has proposed that the antifreeze present in the tissue fluid or muscle-mashed fluid of Nagagashi or Shichirouo, a fish species that has been caught in large quantities in or around Japan and is subject to disposal
  • the present inventors have found a protein, determined its amino acid sequence and gene sequence, and completed the present invention.
  • the present invention is as follows.
  • Antifreeze protein Z-1 represented by the following (a) or (b)
  • Antifreeze protein Z-2 expressed by the following (a) or (b) (a) a protein having the amino acid sequence represented by SEQ ID NO: 4;
  • Antifreeze protein Z-4 expressed by the following (a) or (b)
  • Antifreeze protein Z-5 represented by the following (a) or (b)
  • Antifreeze protein Z-6 represented by the following (a) or (b)
  • Antifreeze protein Z-7 represented by the following (a) or (b)
  • Antifreeze protein Z-8 expressed by the following (a) or (b) (a) a protein having the amino acid sequence represented by SEQ ID NO: 16;
  • Antifreeze protein Z-9 represented by the following (a) or (b)
  • Antifreeze protein Z-10 represented by the following (a) or (b)
  • DNA encoding antifreeze protein Z-3 represented by the following (a) or (b) (a) a protein having the amino acid sequence represented by SEQ ID NO: 6;
  • DNA encoding antifreeze protein Z-9 represented by the following (a) or (b): (a) a protein having the amino acid sequence represented by SEQ ID NO: 18;
  • (23) a nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21, and the antifreeze proteins Z-1, Z-2, Z -DNA encoding 3, Z-4, Z-5, Z-6, Z_7, Z-8, Z-9, Z-10 or Bl.
  • a recombinant vector comprising the DNA according to any one of (12) to (23).
  • a method for producing a protein having an antifreeze function comprising culturing the transformant according to (27) in a medium to produce a protein having an antifreeze function, and collecting the protein. .
  • a method for producing a protein which comprises chemically synthesizing the protein according to any one of (1) to (11) using a peptide synthesizer.
  • a freeze concentration inhibitor comprising the protein according to any one of (1) to (11) as an active ingredient.
  • AFGP antifreeze glycoproteins
  • Antifreeze proteins differ in the amino acid composition of proteins and their higher-order structure, and even if they are classified into the same type, the amino acid sequence and higher-order structure of the protein vary depending on the fish species. (Garth L Fletcher, Choy L Hew, and Peter L Davies (2001) Antifreeze Proteins of Teleost Fishes "Ann. Rev. Physiol. 63, 359-390).
  • ice crystals usually grow into flat hexagonal plates when ice nuclei appear in an aqueous solution. Growth in the direction perpendicular to the plate-like plane is about 100 times slower than growth in the direction of the plate-like plane.
  • AFP antifreeze protein
  • the sample solution will generally contain a pyramid-shaped ice crystal, and crystallographically a hexagonal tetrahedron.
  • a single crystal of ice called a hexagonal pyramid (left figure in Fig. 2) or a bilateral pyramid (left figure in Fig. 2) is observed under a microscope.
  • the presence of AFP results in specific binding of the twelve ice layer planes on the ice crystal to form such a pyramidal ice crystal. This force is macroscopically observed as a non-freezing phenomenon (antifreeze activity) of the specimen.
  • This phenomenon is By using a osmometer, it can be quantified as the freezing point drop or temperature hysteresis of the sample solution.
  • it is necessary to obtain a high-purity aqueous solution of AFP.
  • the antifreeze activity evaluation method by bipyramidal ice crystal observation can be observed if AFP is present (see Choy L. Hew and Daniel SC Yang (1992) "Protein interaction with ice” Eur. J. Biochem. 203, 33-42).
  • the standards were analyzed using a protein sequencer, and the elution time corresponding to each amino acid during separation by HP LC was determined.
  • the lyophilized powder of the antifreeze protein prepared in (1) above was dissolved in acetic acid, and analyzed sequentially from the N-terminal side by a protein sequencer using a general Edman degradation method. Specifically, (i) phenylthiothiocyanate (PITC) was coupled to the N-terminus of the protein to generate phenylthio-powered rubamyl protein (PTC-protein). next, (ii) N-terminal amino acid is cut out from PTC-protein as 2-anilino-5-thiazolinone derivative (ATZ-amino acid) with trifluoroacetic acid.
  • PITC phenylthiothiocyanate
  • the ATZ-amino acid was converted to a thiothiohydantoin derivative (PTH-amino acid) under acidic conditions, separated by HP LC, and the amino acid type was determined based on the elution time.
  • the amino acid sequence was determined by repeating the cycles (i) and (ii) using the remaining protein from which the N-terminal amino acid was cut out as a raw material.
  • a protein having an antifreeze function is included in the present invention.
  • amino acid sequence represented by each SEQ ID NO: ⁇ 7 preferably 1-5, more preferably 1-2 amino acids may be deleted, or 1-7, preferably 1 ⁇ 7 of the amino acid sequence represented by each SEQ ID NO. Five, more preferably one to two amino acids may be added, or the amino acid sequence represented by each SEQ ID NO: Up to 7, preferably 1 to 5, and more preferably 1 to 2 amino acids may be substituted with other amino acids.
  • Nagaji liver was harvested from live fish during the cold season when antifreeze protein expression was increased.
  • the collected liver was cut into 5 mm squares and stored in a 10-fold amount of RNA stabilizer. Liver samples impregnated with the stabilizer were quickly ground at low temperature to extract RNA.
  • MRNA was separated and purified from this RNA solution using an oligo dT matrix. From the purified mRNA, cDNA (1 ststrand) was synthesized with a primer containing oligo dT sequence and reverse transcriptase, and then cDNA (2 ndstrand) was synthesized with RNase H and DNA polimerase I. . Further, both ends of the cDNA were blunted with Pfu DNA Po1 ymerase, and an adapter sequence was added.
  • the amplified genes The fragment was TA cloned.
  • a primer containing an oligo dT sequence For the sequence of the cloned gene fragment, use a primer containing an oligo dT sequence. Determined by the rminator method. The cycle sequence conditions at that time were 96 ° C for 10 seconds, 50 ° C for 5 seconds, 60 ° C for 4 minutes, and 25 cycles.
  • a gene fragment group of about 500 bp which is superior in one of the Nagana-derived cDNA libraries, contains a base sequence encoding an antifreeze protein.
  • a 500 bp cDNA fragment was amplified into a type II, a gene fragment of about 500 bp was amplified using a primer that anneals to the added adapter sequence and a primer containing an oligo dT sequence, and TA cloned.
  • the sequence of the cleaved gene fragment was similarly determined by the Dye Terminator method.
  • the amplified gene fragment was electrophoresed on 0.8% agarose, and a 500 bp long gene fragment was cut out and purified. 20 ng of the purified gene fragment was added to 50 ng of the TA cloning vector, and a ligation reaction was carried out at 4 ° C. with ⁇ T4 DNALigase.
  • the recombinant vector solution was added to the competent cells (DH5 o;) melted on ice, and allowed to stand on ice for 30 minutes. This was heat-treated at 42 ° C for 1 minute, immediately transferred to ice and allowed to stand for 2 minutes. To this, SOC medium was added and incubated at 37 ° C for 30 minutes. The cells were spread on an LB agar medium containing 100 ⁇ g Zm1 ampicillin, and incubated at 37 ° C. for colony formation. An arbitrary colony formed was inoculated into an LB liquid medium containing 100 ⁇ g Zm1 ampicillin, and cultured at 37 ° C. overnight with shaking E. coli. The grown Escherichia coli was destroyed by the alkali-SDS method, and the recombinant vector was separated and purified using a DNA adsorption matrix and a spin column.
  • a base sequence encoding an antifreeze protein was incorporated into a recombinant expression vector containing a promoter, a terminator sequence, etc., and a transformant of this recombinant vector was transformed with 100 ml of Zm1 ampicillin-containing LB liquid medium 10 ml.
  • Add this culture 1 0 m l in 1 00 ⁇ ⁇ / m 1 ampicillin-containing LB liquid medium 1 000m l, was continued further culture.
  • the temperature of the culture was set at 28 ° C.
  • a protein expression inducer was added. Collect the culture solution overnight after the induction of expression, and use the cation exchange solution described in (1).
  • the antifreeze protein was purified using a method by oral chromatography.
  • AFP as a freeze-concentration inhibitor or freezing-point depressant for hydrated products
  • concentration of AFP in the hydrated product is reduced.
  • an amount of at least 0.02% by weight based on the weight of the hydrated substance is added.
  • AFP is most effective when the concentration is within the range of 0.03% by weight to 0.05% by weight.
  • the powder or the aqueous solution of AFP may be mixed as it is.
  • an aqueous solution of AFP may be injected into the hydrate using a syringe or the like.
  • the 5 0-2 Soak hydrate in an aqueous solution of AFP, 0 0 by 0 kg Z cm means such as 2 to pressurize be impregnated with AFP therein hydrous material.
  • the antifreeze protein of the present invention has a function of preventing freezing and concentration of hydrated substances and a function of preventing recrystallization of ice. Regardless of which type of antifreeze protein is classified into AFPI to IV, it can be used as an inhibitor for freeze-concentration of hydrates or as an inhibitor of ice recrystallization, such as in frozen foods or meat Can be used to maintain its quality. This quality maintenance effect can also be expected when cells (egg and sperm), tissues, organs, vegetables, etc. are stored for a long period of time in a frozen state. Further, in recent years, a cold heat supply system or a cold heat storage system using an ice slurry having a large energy density as a heat medium has been proposed.
  • FIG. 1 is a schematic diagram and a photograph showing the shape of a bipyramid ice crystal.
  • the left is a schematic diagram of a hexagonal pyramid
  • the right is a photograph of ice crystals of the hexagonal pyramid observed in Nagaji's blood.
  • Figure 2 is a schematic diagram and a photograph showing the shape of a pyramid-shaped ice crystal. This In the figure, the left shows a schematic diagram of a hexagonal tetrahedron, and the right shows a photograph of ice crystals of the hexagonal tetrahedron observed in blood of Sicily.
  • the fish species used as sources for collecting antifreeze proteins are as follows.
  • Nagagaji Zaarces elongatus kner, English name Notched- Fin eelpout: Caught in Notsuke Bay, Hokkaido.
  • Sicirou (Brachyops is rostratus, Longsnout poacher): Caught in the north sea ⁇ notsuke bay.
  • the temperature in the cooling box in which the sample liquid is set is controlled with an error of +/- 0.1 ° C by the LK600 temperature controller (LK600 temperature controller) manufactured by Linkham.
  • LK600 temperature controller LK600 temperature controller
  • the temperature in the cooling box was lowered at 0.2 ° C per second to minus 22 ° C by the temperature controller.
  • the temperature inside the cooling box was raised to zero. Stop climbing at C and keep it at zero for about 1 to 10 seconds.If it freezes, it melts, passes through countless cracked ice crystal states, and becomes countable ice A single crystal floating in water was observed.
  • the Nagagaji body was cut with a kitchen knife and ground using a mixer.
  • 20 ml of this surimi 20 ml of a 0.1 M aqueous solution of ammonium bicarbonate was added to prepare a 40 ml surimi suspension. This was placed in a plastic test tube and centrifuged at 6,000 rpm for 30 minutes to obtain about 20 ml of a supernatant.
  • the supernatant was subjected to cation exchange chromatography, and the eluate of each lm was collected by a fraction collector while detecting the absorption at 280 nm.
  • the sample solution in which the absorption at 280 nm was observed was purified by reverse phase chromatography using a TO SO HP LC system and an ODS column.
  • the column was equilibrated and the 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 an eluate fraction containing a single protein was obtained and lyophilized.
  • the basic operation relates to the isolation of total RNA was carried out according to the pro-tocol Collection ( "Rneasy R Protect and RNAlater TM Handbook” ⁇ Pi ⁇ RNeasy R Mini Handbook “(QIAGEN)) .
  • RNAlater QIAGEN
  • 60 mg of this liver sample was disrupted in liquid nitrogen, suspended in Buffer RLT 600 1 in 30 mg portions, and further disrupted with QIAshredder (QIAGEN).
  • An equal volume of 70% ethanol was added to the supernatant collected by centrifugation, and the RNA was adsorbed to the column by passing through a spin column. After washing the column with Buffer RW1 and Buffer RPE, RNA was eluted with 30 ⁇ l of RNase free water.
  • mRNA was isolated using 01igotex TM _dT30 and Super> mRNA Purification kit (TaKaRa), and the operation was performed according to the attached protocol.
  • 2xBinding Buffer 150 ⁇ l, Oligotex TM -dT30 and Super> 15 ⁇ l were added to 1.1 / g / 1 total RNA solution 1501, heated at 70 ° C for 3 minutes, and then left at room temperature for 10 minutes. After recovering Oligotex TM -dT30 ⁇ Super> by centrifugation, it was suspended in Wash Buffer and washed with a spin column. The mRNA adsorbed on 01 igotex TM _dT30oku Super> was eluted with 40 ⁇ l of RNase free water heated at 70 ° C.
  • Construction of cDNA library ZAP- cDNA Synthesis Kit c basic operation performed on the (STRATAGENE R) is ⁇ CDNA Synthesis Kit, ZAP - cDNA R Synthesis Kit, and ZAP-cDNA R Gigapack R III Gold Cloning Kit INSTRUCTION MANUAL "( STRATAGENE the recovered mRNA as c above in accordance with R) and ⁇ dNTP containing methylated dCTP (dATP, dCTP, dGTP, the StrataScript RTase used as dTTP) substrate is added, 42 ° C 1 hour reaction
  • the first-strand cDNA was reverse transcribed and synthesized by RNase H and DNA.
  • a degenerate primer was designed based on the amino acid sequence determined by the protein sequencer. Using this primer and a primer complementary to the polyA sequence, a 500 bp cDNA was transformed into a type II Ex Taq TM (TaKaRa) CR (polymerase chain reaction) was performed. The PCR product was cloned into pGEM R -T Easy (Promega), and E. coli was transformed with this plasmid.
  • the transformant is spread on an LB agar medium containing 100 / ig / ml ampicillin, and any colony formed is inoculated into an LB liquid medium containing 100 ⁇ g / ml ampicillin, and shaken at 37 ° C. Culture was continued. Plasmid is separated and purified from the grown E. coli by the alkali-SDS method, and the sequence reaction is performed using the T7 promoter polymer 1 ⁇ and BigDye R Terminator v3.1 and the ycle sequencing Kit (Applied Biosystems), and the ABI PRISM TM The salt line was analyzed using 310 Genetic Analyzer and 3100 Genetic Analyzer (Applied Biosystems).
  • the amino acid sequences of the remaining nine new AFP-Z-2 to Z-10 derived from Nagaagaji were prepared by the same method as described above, and the nucleotide sequences of the CDS regions of those genes were determined by the same method as described above. .
  • the amino acid sequences of the new AFP-Z-2 to Z-10 are shown in SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18 and 20 in the sequence table, and the nucleotide sequences are SEQ ID NOs: 3, 5, 7 , 9, 11, 13, 15, 17 and 19.
  • These nucleotide sequences are the type III antifreeze proteins derived from Macrozoarques americanus whose nucleotide sequences have already been identified. It showed about 75-90% homology with the protein. In this way, new type AFP-Z-2 to Z-10 of type III derived from Nagaji were obtained.
  • RNAlater The basic procedure for isolation of total RNA was in accordance with the protocol collection ("RNeasy R Protect and RNAlater TM Handbook" and RNeasy R Mini Handbook "(QIAGEN)). Collected from live fish during the extremely cold season when the expression of frozen protein is increased .. Collected:! ⁇ 2 g of liver was cut into 5 mm squares and stored in 10 times the amount of RNAlater (QIAGEN). 60 mg was shredded in liquid nitrogen, suspended 30 mg each in Buffer RLT 600 1. After further grinding with QIAshredder (QIAGEN), an equal volume of 70% ethanol was added to the supernatant collected by centrifugation, and the spin column was added. The column was washed with Buffer RW1 and Buffer RPE, and the RNA was eluted with 30 ⁇ l of RNase-free water.
  • mRNA was isolated using 01igotex TM _dT30 ⁇ Super> mRNA Purification kit (TaKaRa), and the operation was performed according to the attached protocol.
  • 2x Binding Buffer 150 ⁇ l, Oligotex TM -dT30 ⁇ Super> 151 was added to 1.1 g / 1 total RNA solution 150 1, and the mixture was heated at 70 ° C for 3 minutes and then left at room temperature for 10 minutes. After recovering 01igotex TM -dT30 ⁇ Super> by centrifugation, the suspension was suspended in Wash Buffer, and ligotex-dT30 Super> was washed with a spin column. Oligotex TM -dT30 and Super mRNA The adsorbed mRNA was eluted with RNase free water 401 heated at 70 ° C. (4) Construction of cDNA library
  • the cDNA library was constructed using the ZAP-cDNA Synthesis Kit (STRATAGENE R ).
  • the basic procedure was "cDNA Synthesis Kit, ZAP-cDNA R Synthesis Kit, and ZAP-cDNA R Gigapack R III Gold Cloning Kit INSTRUCTION MANUAL" R ).
  • dNTP dATP dCTP, dGTP, dTTP
  • StrataScript RTase containing methylated dCTP
  • RNase H, DNA polymerase, and dNTPs were added. C2.
  • the reaction was performed for 5 hours, and second-strand cDNA was synthesized. After blunting the ends of the cDNA with Pfu DNA polymerase, the adapter sequence was ligated with T4 DNA ligase.
  • a degenerate primer is designed based on the amino acid sequence determined by the protein sequencer. Using this primer and a primer complementary to the poly A sequence, the cDNA is transformed into Ex-q type by PCR using Ex Taq TM (TaKaRa). (PCR product was cloned into pGEM R -T Easy (Promega), and E. coli was transformed with this plasmid. The transformant was spread on LB agar medium containing 100 zg / ml ampicillin. The resulting colonies were inoculated into an LB liquid medium containing lOO / zg / ml ampicillin, and cultured by shaking E. coli overnight at 37 ° C. Plasmids were separated from the grown E.
  • PCR polymerase chain reaction
  • the sequence of the fragment was analyzed by the method described above, and as a result, the nucleotide sequence of AFP-B-1 derived from Ciceropodium is as shown in SEQ ID NO: 21.
  • the nucleotide sequence is a type II antifreeze from Lipterus americanus (Semi rter (Hemitripterus americanus)) whose sequence has been determined. It showed about 63% homology with the protein. In this way, the type II antifreeze protein was separated and purified from the surimi of Sicily.
  • 11 types of novel antifreeze proteins were collected from Nagasaki or Shichirouho fish, which are fish caught in the waters near Japan, and their amino acid sequences and nucleotide sequences were determined. Further, it has become possible to produce the antifreeze protein in a large amount by a genetic engineering technique using the obtained gene.
  • these antifreeze proteins can prevent the deterioration of taste and tissue destruction due to the growth of ice crystals in ice cream and frozen foods, for example, and can also prevent the use of ice in a cold heat supply system or cold heat storage using ice slurry. It is expected to be an effective additive that can eliminate the clogging of the piping system due to recrystallization. It is also a promising substance for long-term low-temperature storage of eggs, spermatozoa, and transplanted organs.

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Abstract

L'invention porte sur onze (11) types de protéines antigel trouvées dans des carcasses de poisson des espèces Brachyopsis ou Zoarces vivant dans des rivières du Japon ou des mers proches du Japon ou dans des zones aquatiques de climat similaire à celui du Japon, ainsi que sur leurs séquences d'acides aminés et leurs séquences de base. L'invention porte également sur un procédé de production industrielle de ces protéines antigel utilisant des techniques du génie génétique et les gènes ainsi obtenus et un inhibiteur de concentration du gel ou un dépresseur de point de gélification comprenant ces protéines antigel comme ingrédient actif.
PCT/JP2004/007105 2003-05-21 2004-05-18 Proteine antigel trouvee dans le poisson WO2004104201A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007105734A1 (fr) * 2006-03-13 2007-09-20 Nippon Suisan Kaisha, Ltd. Protéine issue de crustacés présentant une activité antigel
JP2011229504A (ja) * 2010-04-30 2011-11-17 National Institute Of Advanced Industrial Science & Technology 生産性および不凍活性を向上させた改変型不凍タンパク質とその製造方法
EP2596706A4 (fr) * 2010-08-26 2015-01-28 Nichirei Foods Inc Procédé d'augmentation de l'activité d'hystérésis thermique, procédé de réduction de l'inactivation thermique de l'activité d'hystérésis thermique et composition pour l'augmentation de l'activité d'hystérésis thermique

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Publication number Priority date Publication date Assignee Title
JP4748480B2 (ja) 2005-12-09 2011-08-17 独立行政法人産業技術総合研究所 水または含水物の凍結を促進するための材料
JP5171752B2 (ja) * 2009-07-23 2013-03-27 株式会社ノエビア 皮膚外用剤

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HEW C.L. ET AL: "Multiple genes provide the basis for antifreeze protein diversity and dosage in the ocean pout, Macrozoarces Americanus", J. BIOL. CHEM., vol. 263, no. 24, 1988, pages 12049 - 12055, XP002017638 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007105734A1 (fr) * 2006-03-13 2007-09-20 Nippon Suisan Kaisha, Ltd. Protéine issue de crustacés présentant une activité antigel
WO2007105731A1 (fr) * 2006-03-13 2007-09-20 Nippon Suisan Kaisha, Ltd. Protéine présentant un effet de nucléation de la glace
JP2011229504A (ja) * 2010-04-30 2011-11-17 National Institute Of Advanced Industrial Science & Technology 生産性および不凍活性を向上させた改変型不凍タンパク質とその製造方法
EP2596706A4 (fr) * 2010-08-26 2015-01-28 Nichirei Foods Inc Procédé d'augmentation de l'activité d'hystérésis thermique, procédé de réduction de l'inactivation thermique de l'activité d'hystérésis thermique et composition pour l'augmentation de l'activité d'hystérésis thermique

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