JP4446058B2 - Antifreeze protein mixture - Google Patents

Description

  The present invention relates to an antifreeze protein, and a composition containing two or more types of antifreeze proteins having different charges as a whole molecule, and two or more types of antifreeze proteins having different charges as a whole molecule are added. A method for lowering the freezing point of a hydrated product, a method for inhibiting freeze concentration in a hydrated product, a method for inhibiting recrystallization of ice, and a method for storing a hydrated product.

  In a temperature range of 0 ° C. or lower, only water molecules are connected to each other to form an ice lump inside the hydrous material containing water molecules and substances other than water molecules. At this time, a phenomenon called freeze concentration occurs in which substances other than water molecules are forced to move under the physical pressure associated with ice mass formation. When such a hydrate is thawed, the internal structure, physiological activity, flavor, etc. before freezing are impaired. This problem can be solved by combining quick freezing, slow freezing, quick thawing, slow thawing using a conventional program freezer or the like, adding a chemically synthesized substance such as glycerol, or designing the shape of the container for storing the frozen product. It has been overcome by elaborating. However, there are enormous types of target hydrated materials, and there has been a strong demand for the development of a new cryopreservation technique that is more versatile and lower in cost.

  Antifreeze protein (AFP) exhibits properties such as ice recrystallization inhibition and thermal hysteresis activity, and has the ability to strongly suppress freeze-concentration of hydrates. In addition, in the temperature range of 0 ° C. or higher, AFP has the ability to increase the survival rate of the cells by specifically interacting with the cell membrane or water molecules on the cell membrane surface. Because of these capabilities, the addition of AFP preserves the internal structure of various hydrates such as meat, vegetables, processed foods, blood, cells, eggs, sperm, transplanted organs, and consequently the time of the hydrates. It has been considered that it has the effect of preventing deterioration in quality, flavor or life activity associated with the course (storage). For this reason, there has been a strong demand for the development of a method for efficiently producing AFP having the highest preservation effect in food, medicine and other fields at low cost.

  More than 30 years have passed since AFP was discovered in the body fluids of fish inhabiting Antarctica in the late 1960s. In the meantime, enormous research on AFP has been conducted, the possibility of medical application and industrial application of AFP has been examined, and a venture company that produces and sells AFP (A / F Protein Inc. 935 Main Str. Waltham, MA 02451 USA) Established in 1994. Nevertheless, techniques such as low temperature storage by AFP have not been put into practical use. The biggest cause was that a technique for producing the minimum amount of AFP necessary for the practical use of AFP in the medical field, food field and the like has not been developed. Conventionally, the effect of AFP for practical use has been investigated by using AFP purified from fish blood or AFP manufactured by genetic engineering. However, since it is difficult to collect dozens of liters of fish blood using a syringe due to the blood clot phenomenon that occurs after the death of the fish, the amount of AFP purified has been limited (see FIG. 1). In addition, even if a genetic engineering technique is used, it is possible to obtain an amount of about 50 mg in one experiment, and this technique requires a high cost. The amount of AFP required for practical use in the medical field is at least 10 grams, and the amount of AFP required for practical use in the food field is at least 1 kilogram. However, mass production exceeding the gram order of AFP has never been realized, and because of the scarcity of AFP derived from Arctic and Antarctic fish, its selling price has been set at an expensive price that is difficult to put into practical use.

After that, an unlimited amount of AFP can be purified from the surplus of edible muscle species that can be captured without taking a fishing boat in the Arctic Ocean or the Antarctic Ocean without being affected by the death of the fish. (See FIG. 1 and Patent Document 1). At present, attempts by AFP mass refining using this method have already been started by companies and the like, but at the same time, many technical problems have been highlighted. The biggest problem is related to the purification of AFP. AFP possessed by fish is a mixture of AFPs having different activities. If the purification of AFP is continued, it will eventually lead to the separation of 10 or more types of AFP present in the muscle. Each AFP has different biochemical properties and activity as an AFP depending on the amino acid composition. In addition, previous studies have revealed that the survival rate of cells over time varies greatly depending on the AFP used. However, it is not clear what kind of AFP exerts the highest effect, and if the solubility of the selected AFP in water is low, it is not suitable for an additive. Under such circumstances, development of advantageous application conditions of AFP has been desired.
JP 2004-083546 A

  An object of the present invention is to provide an antifreeze protein composition having high antifreeze activity at low cost, and to provide an effective method for applying antifreeze protein at low cost.

  The present inventors have intensively studied to solve the above problems, and as a result of comparing the antifreeze activities of different types of antifreeze proteins, the degree of antifreeze activity varies greatly depending on the charge of the antifreeze proteins. Furthermore, the present inventors have found that an excellent antifreeze activity can be obtained by applying a combination of an antifreeze protein having a negative charge and an antifreeze protein having a positive charge to complete the present invention.

That is, the present invention includes the following inventions.
(1) A composition comprising a positively charged antifreeze protein and a negatively charged antifreeze protein as active ingredients.
(2) The composition according to (1), comprising a positively charged antifreeze protein and a negatively charged antifreeze protein in a molar ratio of 99: 1 to 1:99.
(3) The positively charged antifreeze proteins are the following proteins (a) and (b):
(A) a protein comprising the amino acid sequence represented by any of SEQ ID NOs: 2, 4, 6, 8, 10 or 12,
(B) an amino acid sequence represented by any one of SEQ ID NOs: 2, 4, 6, 8, 10 or 12, comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and antifreeze At least one selected from proteins having activity;
The negatively charged antifreeze proteins are the following proteins (c) and (d):
(C) a protein comprising the amino acid sequence represented by any of SEQ ID NOs: 14, 16, 18, 20, 22, 24 or 26,
(D) an amino acid sequence represented by any one of SEQ ID NOs: 14, 16, 18, 20, 22, 24 or 26, comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added; and The composition according to (1) or (2), which is at least one selected from proteins having antifreeze activity.

(4) The composition according to any one of (1) to (3), for lowering the freezing point of the hydrous material.
(5) The composition according to any one of (1) to (3), which inhibits freeze concentration in a hydrated product.
(6) The composition according to any one of (1) to (3), which inhibits recrystallization of ice in a hydrous material.
(7) The composition according to any one of (1) to (3), for storing a hydrated product.
(8) A method for lowering the freezing point of a hydrous material, the method comprising adding a positively charged antifreeze protein and a negatively charged antifreeze protein to the hydrous material.
(9) A method for inhibiting freeze concentration in a hydrated product, the method comprising adding a positively charged antifreeze protein and a negatively charged antifreeze protein to the hydrated product.
(10) A method for inhibiting recrystallization of ice in a hydrous material, the method comprising adding a positively charged antifreeze protein and a negatively charged antifreeze protein to the hydrous material .
(11) A method for preserving a hydrated product, comprising adding a positively charged antifreeze protein and a negatively charged antifreeze protein to the hydrated product.
(12) The positively charged antifreeze protein and the negatively charged antifreeze protein are added at a molar ratio of 99: 1 to 1:99, according to any one of (8) to (11) the method of.

(13) The positively charged antifreeze proteins are the following proteins (a) and (b):
(A) a protein comprising the amino acid sequence represented by any of SEQ ID NOs: 2, 4, 6, 8, 10 or 12,
(B) an amino acid sequence represented by any one of SEQ ID NOs: 2, 4, 6, 8, 10 or 12, comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and antifreeze At least one selected from proteins having activity;
The negatively charged antifreeze proteins are the following proteins (c) and (d):
(C) a protein comprising the amino acid sequence represented by any of SEQ ID NOs: 14, 16, 18, 20, 22, 24 or 26,
(D) an amino acid sequence represented by any one of SEQ ID NOs: 14, 16, 18, 20, 22, 24 or 26, comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added; and The method according to any one of (8) to (12), which is at least one selected from proteins having antifreeze activity.

  The present invention provides an antifreeze protein composition having high antifreeze activity at low cost, and an effective method for applying antifreeze protein at low cost.

  In the present invention, antifreeze protein (AFP) means a protein having antifreeze activity, and grows by binding specifically to the surface of ice crystals generated in cells in a freezing temperature range in a living body such as fish. It is known as a biological defense substance that suppresses and protects the body from freezing of tissues. In this specification, antifreeze protein includes both peptides and proteins having antifreeze activity.

  The antifreeze activity of the antifreeze protein is evaluated as an activity that brings any of thermal hysteresis, ice crystal growth inhibition and ice recrystallization inhibition to a hydrate containing the protein. Alternatively, characteristically shaped ice crystals (for example, bipyramidal ice crystals with two pyramids stacked on the bottom surface for fish-derived antifreeze proteins) are formed in water containing antifreeze proteins. Therefore, the antifreeze activity of the target protein can also be evaluated by observing the shape of ice crystals with a microscope.

  Normally, the freezing point of water and the melting point of ice are the same. However, if antifreeze protein is present, it binds to ice crystals, so that there is a difference between the freezing point of water and the melting point of ice. This phenomenon is called thermal hysteresis. The magnitude of antifreeze activity in antifreeze protein is usually evaluated by the difference between the melting point of ice and the freezing point of water that occurs when antifreeze protein is present. In this specification, the difference between the melting point and the freezing point is referred to as thermal hysteresis. Define as active. Thermal hysteresis activity can be measured by using an osmometer (osmometer). In addition, the formed ice crystals grow by absorbing water generated by sublimation or partial melting at a relatively high temperature of −10 ° C. or higher. Ice recrystallization inhibition refers to the effect of inhibiting this phenomenon.

  In the present invention, the hydrated product means a substance containing water molecules and molecules other than water molecules, for example, an aqueous solution composed of a solute and a solvent, a mixed solution of a substance that does not dissolve in water and water, cereals, noodles, eggs Foods such as vegetables, fruits, meat, seafood, bread dough, ice confectionery and processed foods, medical products, diagnostics, reagents, cosmetics, lotions, blood, serum, platelets, sperm, eggs, single cells, multi-cells, living organisms Examples thereof include tissues such as tissues, heart, pancreas, liver and kidney, and preservation solutions thereof, snow melting agents, frost damage prevention agents and the like.

  Among the antifreeze proteins of different types, the present inventors have shown that negatively charged antifreeze proteins have high antifreeze activity, whereas positively charged antifreeze proteins have almost no antifreeze activity. I found that I do not have. Furthermore, high antifreeze activity can be obtained by adding a small amount of negatively charged antifreeze protein to a positively charged antifreeze protein that has almost no antifreeze activity. It was found that the antifreeze activity far superior to that of the antifreeze protein alone was obtained. Therefore, according to the present invention, it is possible to save negatively charged antifreeze proteins having high activity alone, and it is possible to use all types of antifreeze proteins without waste. As a result, it greatly contributes to the practical application of antifreeze proteins, and is very advantageous in terms of cost.

  In one embodiment, the present invention relates to a composition comprising a positively charged antifreeze protein and a negatively charged antifreeze protein as active ingredients.

  In the present invention, a positively charged antifreeze protein means a non-binding protein that binds to a cation exchange resin, in particular, an ion exchange resin having a sulfopropyl (SP) group as a charged group. It means frozen protein. In addition, negatively charged antifreeze proteins have negatively charged molecular surfaces and bind to anion exchange resins, particularly ion exchange resins having quaternary aminoethyl (QAE) groups as charged groups. Means antifreeze protein.

  Each of the positively charged antifreeze protein and the negatively charged antifreeze protein may include one type of antifreeze protein, or may include two or more types of antifreeze proteins.

  In the composition of the present invention, the ratio of the positively charged antifreeze protein to the negatively charged antifreeze protein is a molar ratio, preferably 99: 1 to 1:99, more preferably 95: 5. -50: 50, more preferably 90: 10-80: 20. By blending or adding antifreeze proteins having different charges in the above ratio, a much higher antifreeze activity, particularly thermal hysteresis activity, can be obtained than when each is used alone. In addition, a high amount of antifreeze protein can be obtained by adding a small amount of highly active antifreeze protein to an antifreeze protein having almost no activity. The charged antifreeze protein can be used effectively, and the negatively charged antifreeze protein with a relatively small amount but high activity can also be used efficiently.

  In the present invention, the positively charged antifreeze protein and the negatively charged antifreeze protein may be synthetic or naturally derived. Although it does not specifically limit as natural antifreeze protein, For example, antifreeze protein derived from fish, insect origin, fungi origin, mammal origin, and plant origin is mentioned. Antifreeze protein derived from fish is preferred, more preferred is a fish of the genus Zoarces, and particularly preferred is an antifreeze protein obtained from muscles of Nagareji (Zoarces elongatus Kner).

  The Hokkaido Notsuke Fishery Cooperative in 179-2 Owanuma Port Town, Betsukai-cho, Notsuke-gun, Hokkaido, is engaged in industrial development centered on the North Sea striped shrimp, etc. captured in Notsuke Bay. It has become a problem to eat. Nagagaji is caught in a large amount of nets along with the North Sea striped shrimp during fishing using a bottom net, and is separated and collected into a basket for discarded fish and discarded by a disposal company. Therefore, Nagagaji is free of cost, and is a very advantageous fish species for keeping it at a low price as a raw material for producing antifreeze protein.

  There are mainly four types of fish-derived antifreeze proteins: AFPI with a molecular weight of about 3,000 to 4,500, AFPII with a molecular weight of about 20,000, AFPIII with a molecular weight of about 7,000, and AFPIV with a molecular weight of about 11,000. It is classified. A preferred antifreeze protein in the present invention is AFPIII.

  In the present invention, antifreeze proteins derived from different sources may be combined. For example, in the case of antifreeze proteins derived from fish, AFPI to AFPIV may be combined. Preferably, antifreeze proteins derived from the same source, more preferably the same type of antifreeze protein among AFPI to AFPIV, and more preferably the same type of AFP isoform are combined.

  Preferable examples of the positively charged antifreeze protein include a protein having an amino acid sequence represented by any one of SEQ ID NOs: 2, 4, 6, 8, 10 or 12. A protein consisting of the amino acid sequence represented by SEQ ID NO: 4 or 12 is more preferred, and a protein consisting of the amino acid sequence represented by SEQ ID NO: 12 is particularly preferred.

  Preferable examples of the negatively charged antifreeze protein include a protein having an amino acid sequence represented by any of SEQ ID NOs: 14, 16, 18, 20, 22, 24, or 26. These proteins are based on amino acid sequence similarity, a group of proteins consisting of the amino acid sequence of SEQ ID NO: 14, 16, 18 or 20 (QAE1 group) and a group of proteins consisting of the amino acid sequence of SEQ ID NO: 22, 24 or 26 ( QAE2 group).

  The QAE1 group and the QAE2 group are classified based on the difference in amino acid residues that are mainly observed on the N-terminal side of the molecule (in FIG. 3, 2, 9, 19, 20, 23-25, 27, 30). And amino acid residue # 41). In addition, the isoelectric point of the antifreeze protein of the QAE1 group is in the range of 8-9, whereas the isoelectric point of the antifreeze protein of the QAE2 group is in the range of 4-7. Both exhibit different antifreeze activities while having the sequence characteristics of the QAE group.

  In the present invention, a protein belonging to the QAE1 group is preferable, and a protein consisting of the amino acid sequence represented by SEQ ID NO: 16 is particularly preferable.

  In the present invention, a composition comprising, as active ingredients, a protein comprising the amino acid sequence represented by SEQ ID NO: 12 and a protein comprising the amino acid sequence represented by SEQ ID NO: 16 is particularly preferred.

  Proteins functionally equivalent to the above-mentioned amino acid sequences can also be used as preferred antifreeze proteins. Functionally equivalent means that the target protein has the same biological function, biochemical function, and antifreeze activity as the protein consisting of each amino acid sequence.

  A protein functionally equivalent to a protein consisting of each amino acid sequence includes a protein containing each amino acid sequence, and an amino acid sequence in which one or several amino acids are deleted, substituted or added in each amino acid sequence, and Proteins having antifreeze activity are included. Here, the term “several” means usually 2 to 10, preferably 2 to 5, more preferably 2 to 3. The amino acid chain length of a protein containing each amino acid sequence and having antifreeze activity is usually 10 to 200 residues, preferably 20 to 180 residues, more preferably 35 to 140 residues.

  Also, each amino acid sequence has at least 50% identity, preferably 75% identity, more preferably 85% identity, more preferably 90% identity, particularly preferably 95% identity. Proteins containing amino acid sequences with identity and having antifreeze activity are also included.

  Amino acid sequence identity can be determined by methods commonly used in the art, such as determined by the algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993). can do. Based on this algorithm, programs called BLASTN and BLASTX have been developed (Altschul et al. J. Mol. Biol. 215: 403-410, 1990). Specific techniques for these analysis methods are known (http://www.ncbi.nlm.nih.gov.).

  In the present invention, the positively charged antifreeze protein and the negatively charged antifreeze protein can be obtained from natural sources such as fish, for example, blood and muscle of fish, by a technique known in the art. it can.

  When obtaining antifreeze protein from the blood of fish, the blood collected from the tail vein of the AFP-producing fish using a syringe is allowed to stand at around 4 ° C. for 12 hours or more. The blood cell component (without antifreeze protein) precipitated by this operation is gently removed (decantation) so as not to break the red blood cell membrane. By applying a general-purpose biochemical separation operation such as dialysis, ion exchange chromatography or HPLC chromatography to the remaining serum components (including antifreeze protein), the antifreeze protein can be obtained (Schrag, J D., et al., Biochim.Biophys. Acta, 915, 357-370 (1987)).

  When obtaining antifreeze protein from fish muscle, the AFP-producing fish body is used as it is, or the fish body with its head and internal organs removed is ground into a surimi. Add water or buffer to this and suspend well. At this time, the surimi suspension is heated at 50 to 90 ° C. as necessary (eg, when purifying type I and type AFP). The surimi suspension is allowed to stand or centrifuged at 3,000 to 12,000 rpm for about 30 minutes to obtain a supernatant. On the other hand, an antifreeze protein can be obtained by applying a general-purpose biochemical separation operation such as dialysis, ion exchange chromatography or HPLC chromatography (Japanese Patent Laid-Open No. 2004-083546).

  Moreover, it can synthesize | combine by the method well-known in this technical field, for example, a solid-phase peptide synthesis method, etc. using the said amino acid sequence or the publicly available amino acid sequence information. It is also possible to produce an antifreeze protein using information on cDNA encoding the antifreeze protein using a known genetic recombination technique.

  The base sequence of the DNA encoding the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 is shown in SEQ ID NO: 1, and the base sequence of the DNA encoding the protein consisting of the amino acid sequence represented by SEQ ID NO: 4 is shown in SEQ ID NO: 3. The base sequence of DNA encoding the protein consisting of the amino acid sequence represented by SEQ ID NO: 6 is shown in SEQ ID NO: 5, and the base sequence of DNA encoding the protein consisting of the amino acid sequence represented by SEQ ID NO: 8 is SEQ ID NO: 7. The base sequence of DNA encoding the protein consisting of the amino acid sequence represented by SEQ ID NO: 10 is shown in SEQ ID NO: 9, and the base sequence of DNA encoding the protein consisting of the amino acid sequence represented by SEQ ID NO: 12 is SEQ ID NO: 11. The nucleotide sequence of DNA encoding the protein consisting of the amino acid sequence represented by SEQ ID NO: 14 is 3, the nucleotide sequence of the DNA encoding the protein consisting of the amino acid sequence represented by SEQ ID NO: 16 is shown in SEQ ID NO: 15, and the nucleotide sequence of the DNA encoding the protein consisting of the amino acid sequence represented by SEQ ID NO: 18 is SEQ ID NO: 17, the nucleotide sequence of the DNA encoding the protein consisting of the amino acid sequence represented by SEQ ID NO: 20 is shown in SEQ ID NO: 19, and the nucleotide sequence of the DNA encoding the protein consisting of the amino acid sequence represented by SEQ ID NO: 22 is SEQ ID NO: 21 shows the base sequence of the DNA encoding the protein consisting of the amino acid sequence represented by SEQ ID NO: 24, and SEQ ID NO: 23 shows the base sequence of the DNA encoding the protein consisting of the amino acid sequence represented by SEQ ID NO: 26. 25, respectively.

Hereinafter, production of antifreeze protein using a recombinant technique will be described.
The recombinant vector for producing antifreeze protein can be obtained by ligating the above DNA base sequence or the publicly available cDNA base sequence to an appropriate vector. The vector can be obtained by introducing it into a host so that the antifreeze protein can be expressed.

  As the vector, a phage or a plasmid capable of autonomously growing in a host microorganism is used. Plasmid DNA includes plasmids derived from E. coli (eg, pET21a, pGEX4T, pUC118, pUC119, pUC18, pUC19, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, etc.), yeast-derived plasmids (eg, YEp13, YEp24, YCp50, etc.) ) And the like, and examples of the phage DNA include λ phage (λgt11, λZAP, etc.). Furthermore, animal viruses such as vaccinia virus and insect virus vectors such as baculovirus can be used.

  In order to insert the antifreeze protein cDNA into a vector, first, the purified DNA is cleaved with an appropriate restriction enzyme, inserted into a restriction enzyme site of a suitable vector DNA or a multicloning site, and then linked to the vector. Adopted.

  In addition, recombinant vectors for producing antifreeze proteins used in mammalian cells include promoters, antifreeze protein cDNAs, cis elements such as enhancers, splicing signals, poly A addition signals, selectable markers, ribosome binding sequences, as desired. (SD sequence) or the like may be linked.

  A known DNA ligase is used to link the DNA fragment and the vector fragment. Then, the DNA fragment and the vector fragment are annealed and then ligated to produce a recombinant vector for producing antifreeze protein.

  The host used for transformation is not particularly limited as long as it can express the antifreeze protein. Examples include bacteria (E. coli, Bacillus subtilis, etc.), yeast, animal cells (COS cells, CHO cells, etc.) and insect cells.

  For example, when a bacterium is used as a host, an antifreeze protein-producing recombinant vector is autonomously replicable in the bacterium, and at the same time, it is composed of a promoter, a ribosome binding sequence, an antifreeze protein DNA, and a transcription termination sequence. Preferably it is. Moreover, the gene which controls a promoter may be contained. Examples of E. coli include Escherichia coli BRL. Examples of Bacillus subtilis include Bacillus subtilis. Any promoter can be used as long as it can be expressed in a host such as Escherichia coli. The method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. Examples thereof include a method using calcium ions and an electroporation method.

  When yeast, animal cells, insect cells and the like are used as hosts, antifreeze proteins can be similarly produced according to techniques known in the art.

  Antifreeze protein can be obtained by culturing the transformant prepared above and collecting it from the culture. “Culture” means any of culture supernatant, cultured cells, cultured cells, or disrupted cells or cells. The method of culturing the transformant in a medium is performed according to a usual method used for host culture.

  As a medium for culturing transformants obtained using microorganisms such as Escherichia coli and yeast as a host, the medium contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the microorganisms. As long as the medium can be used, either a natural medium or a synthetic medium may be used.

  The culture is usually performed at 37 ° C. for 6 to 24 hours under aerobic conditions such as shaking culture or aeration and agitation culture. During the culture period, the pH is kept near neutral. The pH is adjusted using an inorganic or organic acid, an alkaline solution, or the like. During culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary.

  When antifreeze protein is produced in cells or cells after culturing, the protein is extracted by disrupting the cells or cells. When antifreeze protein is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. Thereafter, by using general biochemical methods used for protein isolation and purification, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc. alone or in appropriate combination, Antifreeze protein can be isolated and purified from

  Whether or not antifreeze protein is obtained can be confirmed by SDS-polyacrylamide gel electrophoresis or the like.

  The recombinant antifreeze protein obtained by the above method includes a fusion protein with any other protein. Examples thereof include a fusion protein with glutathione-S-transferase (GST) and green fluorescent protein (GFP). Furthermore, the peptide expressed in the transformed cell may be subjected to various modifications in the cell after being translated. Therefore, modified peptides can also be used as antifreeze proteins. Examples of such post-translational modifications include elimination of N-terminal methionine, N-terminal acetylation, sugar chain addition, limited degradation by intracellular protease, myristoylation, isoprenylation, phosphorylation and the like.

  The composition of the present invention includes a positively charged antifreeze protein and a negatively charged antifreeze protein, but not only a composition obtained by mixing each purified protein, An antifreeze protein mixture is also included as a crude product containing both antifreeze proteins obtained in the step of purifying materials such as fish.

  Such an antifreeze protein mixture can be prepared, for example, by preparing a fish surimi or dried fish suspension and leaving or suspending the fish surimi or dried fish suspension to obtain a supernatant. In response, the supernatant is heat-treated, and the resulting precipitate is removed by centrifugation to obtain a supernatant containing antifreeze protein, and the antifreeze protein is recovered from this supernatant.

  More specifically, the fish meat is ground, or the dried fish is finely cut with scissors and then pulverized with a mixer, etc., and then an aqueous solution such as water, ammonium bicarbonate or sodium hydrogen phosphate is added thereto to suspend the fish meat. Use a suspension. 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. The conditions for the centrifugation of the surimi suspension or the dried fish ground product suspension are 3,000 to 12,000 rpm and 5 to 60 minutes. In particular, when purifying type I and type II antifreeze proteins, heat treatment of the resulting supernatant effectively removes or reduces fish and surimi-specific odors. Miscellaneous proteins can be heat denatured and precipitated. This heat treatment is usually performed at 30 to 90 ° C, more preferably at 50 to 75 ° C, and usually for 1 to 90 minutes, preferably 20 to 40 minutes. When the heat treatment is not performed, the odor peculiar to fish and surimi can be removed or reduced by passing through an activated carbon filter. Subsequently, the obtained supernatant is centrifuged to remove precipitated contaminating proteins. The obtained supernatant containing the antifreeze protein at a high concentration may be used as it is, but is preferably made into a dry powder by lyophilization (Japanese Patent Laid-Open No. 2004-83546).

  Since the composition of the present invention has a high antifreeze activity, it can be used as a composition for lowering the freezing point of a hydrated product, particularly an aqueous solution. In addition, the composition of the present invention can be used as a composition for inhibiting freeze concentration in a hydrated product. Furthermore, the composition of the present invention can be used as a composition for inhibiting recrystallization in a hydrated product. Antifreeze protein, particularly antifreeze protein having thermal hysteresis activity, is known to have an action of inhibiting freeze concentration and maintaining its quality and physiological activity when added to a hydrate (Japanese Patent Application No. 2003-78977 and Rubinsky, B., et al., Biochem. Biophys. Res. Commun., 180 (2) 566-571 (1991)). Therefore, the composition of the present invention is a hydrated product, in particular, foods such as cereals, noodles, eggs, vegetables, fruits, meat, seafood, bread dough, ice confectionery and processed foods, medical products, diagnostic agents, reagents, cosmetics, Lotion, blood, serum, platelets, sperm, ovum, single cell, multi-cell, living tissue, heart, pancreas, liver, kidney and other organs, and their preservatives, snow melting agents, frost damage prevention agents, etc., preferably at low temperature For example, it can be used as a composition for storage at -30 to + 10 ° C. By using the composition of the present invention for storage, the quality of the hydrated product can be maintained. Moreover, the deactivation of a physiologically active substance can be suppressed. Here, as the physiologically active substance, in particular, substances derived from living bodies, such as antibiotics, functional food additives, cell differentiation inducers, functional lipids (DHA, EPA, etc.), low molecular compounds, hormones, vitamins, peptides , Enzymes, antibodies, proteins and the like. Examples of the hydrous substance containing a physiologically active substance include aqueous solutions containing physiologically active substances, biological cells, tissues, organs, body fluids such as blood, microorganisms such as bacteria and viruses.

  The composition of the present invention may contain salts, ions, sugars, nucleic acids, low molecular weight substances, high molecular weight substances, peptides, muscle-derived components and the like in addition to antifreeze proteins.

  The present invention also provides a method for lowering the freezing point of a hydrated product, particularly an aqueous solution, comprising adding a positively charged antifreeze protein and a negatively charged antifreeze protein, and freeze concentration in a hydrated product. The present invention relates to a method for inhibiting, a method for inhibiting recrystallization of ice in a water-containing material, and a method for storing a water-containing material. In these methods, the addition ratio of the positively charged antifreeze protein and the negatively charged antifreeze protein is the same as in the above-described composition of the present invention. The total amount of antifreeze protein added to the water-containing material is usually 0.0001 to 1.0% by mass, preferably 0.0001 to 0.01% by mass, more preferably 0.0001 to 0.001% by mass. is there. Moreover, in order to preserve hydrated products such as foods, cells and organs, it is preferable to immerse the desired hydrated product in an aqueous solution (preservation solution) containing antifreeze proteins. In such a case, the concentration of the antifreeze protein in the aqueous solution is usually 0.0001 to 10.0% by mass, preferably 0.001 to 1.0% by mass, more preferably 0.005 to 0.05% by mass. %. In the method of the present invention, the positively charged antifreeze protein and the negatively charged antifreeze protein may be added simultaneously or separately. Alternatively, an antifreeze protein mixture as a crude product containing both antifreeze proteins obtained at the stage of purifying materials such as fish may be added.

Example 1
(1) Specimen sample The fish species used as the source of antifreeze protein are as follows.
Nagareji (Zoarces elongatus kner, English name: Notched-Fineelpout): caught in Notsuke Bay, Hokkaido.

(2) Determination of amino acid sequence of Nagagaji AFP Nagagaji was cut with a knife and ground using a mixer. By adding 20 ml of 0.1 M aqueous ammonium bicarbonate to 20 ml of this surimi, 40 ml of surimi suspension was prepared. This was put in a plastic test tube and centrifuged at 6,000 rpm for 30 minutes to obtain about 20 ml of supernatant. The 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. For cation exchange chromatography, an Amersham Pharmacia Biotech FPLC system and a BIO-RAD High-S column were used. 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. went. All the operations so far were performed at 4 ° 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. The elution pattern in this HPLC operation is shown in the upper part of FIG. The 14 fractions thus obtained were dissolved in 0.1M ammonium bicarbonate aqueous solution and their bipyramidal ice crystals were observed. The results are shown in the lower part of FIG. As a result, bipyramidal ice crystals demonstrating the antifreeze activity of the AFP isoform were observed in 13 fractions from 2 to 14. Thus, after purifying samples of 13 types of Nagagadi AFP, amino acid sequences were determined using samples obtained by freeze-drying them. The dried sample was dissolved in acetic acid and added to a polybrene-treated cartridge filter. Edman degradation was performed using a 491 protein sequencer manufactured by Applied Biosystems, and amino acid sequences were sequentially determined from the N-terminal side.

(3) Isolation of total RNA Basic procedures for isolation of total RNA follow protocol collections ("RNeasy (registered trademark) Protect and RNAlater ^™ Handbook" and "RNeasy (registered trademark) Mini Handbook" (QIAGEN)). It was. The Nagagaji liver as a sample was collected from raw fish during the severe cold season when the expression level of antifreeze protein increased. The collected 1 to 2 g of liver was cut into 5 mm square and stored in 10 times amount of RNAlater (QIAGEN). 60 mg of this liver sample was pulverized in liquid nitrogen, 30 mg each was suspended in 600 μl of Buffer RLT, and further pulverized with QIAshredder (QIAGEN). An equal amount of 70% ethanol was added to the supernatant collected by centrifugation, and RNA was adsorbed onto the column by passing through a spin column. After washing the column with Buffer RW1 and Buffer RPE, RNA was eluted with 30 μl of RNase-free water.

(4) Isolation of mRNA mRNA was isolated using Oligotex ^™ -dT30 <Super> mRNA Purification kit (TaKaRa), and the operation was performed according to the attached protocol. To 150 μl of 1.1 μg / μl total RNA solution, 150 μl of 2 × binding buffer and Oligotex ^™ -dT30 <Super> 15 μl were added, heated at 70 ° C. for 3 minutes, and then allowed to stand at room temperature for 10 minutes. Oligotex ^™ -dT30 <Super> was collected by centrifugation and then suspended in a washing buffer, and Oligotex ^™ -dT30 <Super> was washed with a spin column. The mRNA adsorbed on Oligotex ^™ -dT30 <Super> was eluted with 40 μl of RNase-free water heated at 70 ° C.

(5) Construction of cDNA library The construction of the cDNA library was carried out with ZAP-cDNA Synthesis Kit (STRATAGENE (registered trademark)). The basic operation was in accordance with “cDNA Synthesis Kit, ZAP-cDNA (registered trademark) Synthesis Kit, and ZAP-cDNA (registered trademark) Gigapack (registered trademark) III Gold Cloning Kit INSTRUTION MANUAL” (STRATAGENE). Using the mRNA recovered as described above as a template, using dNTP containing methylated dCTP (dATP, dCTP, dGTP, dTTP) as a substrate, adding StrataScript RTase, and reacting at 42 ° C. for 1 hour, the first reaction is performed. Strand cDNA was reverse transcriptionally synthesized. Next, RNase H, DNA polymerase, and dNTP were added and reacted at 16 ° C. for 2.5 hours to synthesize second strand cDNA. After smoothing the ends of the cDNA with Pfu DNA polymerase, the adapter sequence was ligated with T4 DNA ligase.

(6) Determination of base sequence When a part of the constructed cDNA library was electrophoresed with 0.8% agarose, a gene fragment of 500 bp was mainly confirmed. Therefore, a degenerate primer was designed based on the amino acid sequence determined by the protein sequencer, and PCR was performed with ExTaq ^™ (TaKaRa) using this primer and a primer complementary to the polyA sequence and a 500 bp cDNA as a template. The PCR product was cloned into pGEM®-T Easy (Promega) and E. coli was transformed with this plasmid. The transformant was spread on an LB agar medium containing 100 μg / ml ampicillin, an arbitrary colony formed was inoculated into an LB liquid medium containing 100 μg / ml ampicillin, and Escherichia coli was cultured overnight at 37 ° C. with shaking. Plasmids were separated and purified from the grown Escherichia coli by alkali-SDS method, and sequence reaction was performed using T7 promoter primer and BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), and ABI PRISM ^TM 310 Genetic Gene The base sequence was analyzed by 3100 Genetic Analyzer (Applied Biosystems). From this analysis result, it was confirmed that the 500 bp cDNA contained a base sequence group encoding an amino acid sequence having high homology with the separated and purified antifreeze protein. Thus, PCR was performed with ExTaq ^™ (TaKaRa) using the 500 bp cDNA as a template and a primer that anneals to the linker sequence and poly A sequence to amplify the entire base sequence encoding the antifreeze protein. These PCR products were cloned into pGEM (registered trademark) -T Easy (Promega), and the sequence of the gene fragment was analyzed by the method described above.

  About 13 types of AFP (nfeAFP1-13) derived from Nagagaji, their amino acid sequences are represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, respectively. The base sequence of the CDS region is shown in 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25. Moreover, the table | surface which classified and summarized these amino acid sequences is shown in FIG. These AFPs include AFPIII purified from blood of Macrozoarces americanus (Hew, CL, et al., J. Biol. Chem. 263 (24), 12049-12055 (1988)). Since it showed 75-90% homology, it is classified as AFPIII. In FIG. 3, nfeAFP represents an abbreviation obtained by combining AFP with the initial letter nfe of Notched-fin elepout, which is the scientific name of Nagagaji, and nfeAFP1 to 13 represent 13 types of Nagagaji-derived AFP isoforms. Gray shaded portions in FIG. 3 show amino acid sequences that are homologous to each other. For isoforms in which lysine (K) is observed at the C-terminus of the amino acid sequence shown in FIG. 3, it is assumed that this C-terminal lysine is cleaved and deleted after translation. The above Hew, C.I. L. Based on the similarity comparison between the amino acid sequences of the isoforms classified in, et al. And the amino acid sequences other than the gray hatched portion in FIG. 3, this AFP isoform has a positively charged molecular surface. Antifreeze protein group (SP group) that binds to cation exchange resin (SP-Sephadex) and antifreeze protein group (QAE group) that binds to anion exchange resin (QAE-Sephadex) with negatively charged molecular surface ). Further, the seven types of isoforms belonging to the QAE group were classified into these two types of groups based on the characteristic differences in the shaded gray areas shown for the QAE1 and QAE2 groups in FIG. These 13 types of AFP isoforms are considered to roughly correspond to 13 HPLC fractions (upper part of FIG. 2) obtained from muscle as an AFP final purified product.

Example 2
In order to compare the antifreeze activity of five types of AFP isoforms (nfeAFP2, 6, 8, 11, 13) whose sequences are shown in FIG. 3, they were expressed by genetic engineering. Using a PCR method, a fragment in which NdeI and XhoI restriction enzyme sites were added to both ends of DNA encoding each isoform was amplified and then incorporated into a pET vector (Novagen) digested with the same restriction enzyme. Escherichia coli BL21 (DE3) was transformed with each expression vector, spread on LB agar medium containing ampicillin, and cultured at 37 ° C. The formed colonies were inoculated into LB liquid medium containing 100 μg / ml of ampicillin and cultured with shaking at 28 ° C. overnight. The next day, the cells were transferred to LB liquid medium containing 100 μg / ml of ampicillin and cultured at 28 ° C. The degree of growth of E. coli was estimated from turbidity. D. The expression of AFP was induced by adding isopropyl-β-D-thiogalactopyranoside at a final concentration of 0.5 mM to a logarithmic growth machine of 600 = 0.5. After overnight shaking culture, the cells were centrifuged at 6,000 rpm for 30 minutes to recover E. coli and sonicated in a 10 mM Tris-HCL / 1 mM EDTA solution. The supernatant was collected by centrifugation at 11,000 rpm for 30 minutes, dialyzed against 50 mM sodium citrate buffer (pH = 2.9) for nfeAFP11, and 50 mM sodium acetate buffer (pH = 3.7) for the others. Covalent proteins were aggregated. This was removed by centrifugation at 11,000 rpm for 30 minutes to obtain a supernatant. The supernatant was subjected to cation exchange chromatography, and 2 ml of the eluate was collected by a fraction collector while detecting absorption at 280 nm. Bio-Rad Duo Flow system and High-S column were used for cation exchange chromatography. The column was equilibrated and the supernatant was taken up using 50 mM sodium citrate buffer (pH = 2.9) or 50 mM sodium acetate buffer (pH = 3.7), and the elution was 0 at a flow rate of 1 ml / min. This was done by applying a linear gradient of ~ 0.5M sodium chloride. In this way, five types of AFP isoforms were purified.

Example 3
The five types of AFP isoforms (nfeAFP2, 6, 8, 11, 13) purified in Example 2 were each dialyzed with 0.1 M aqueous ammonium bicarbonate solution (pH = 7.9) to the desired concentration. After that, the protein concentration dependence of the thermal hysteresis activity was measured for each. An osmometer (VOGEL Osmometer) was used for the freezing point measurement, and a low temperature microscope (Leica DMLB 100 type microscope and Linkam LK600 type cooling device) with a thermistor thermometer was used for the melting point measurement. The difference between the obtained freezing point and the melting point was defined as thermal hysteresis activity. The value of thermal hysteresis activity reflects the antifreeze activity of each AFP isoform, ie the ability to bind ice crystals. The result of this measurement is shown in FIG. The molar concentration of AFP isoform at each measurement point was determined by measuring the absorbance of the aqueous solution.

  As for nfeAFP13 containing cysteine in the primary sequence, since formation of multimers was confirmed in the purified sample, it was monomerized with the monomerized state (+ DTT) by adding a reducing agent (dithiothreitol). Thermal hysteresis activity was measured for both the absence state (-DTT). Since the concentration is determined by absorbance, any of these measured values can be regarded as thermal hysteresis activity per mole of monomer of nfeAFP13.

  From FIG. 4, it was clarified that the value of thermal hysteresis activity differs greatly among the five types of AFP isoforms. This indicates that the antifreeze activity varies greatly depending on the type of AFP isoform, that is, the charge possessed by the AFP isoform. In particular, AFP isoforms (nfeAFP2, 6) belonging to the SP group were shown to have zero antifreeze activity by themselves. Further, it was found that even AFP isoforms belonging to the QAE group have a large difference in antifreeze activity depending on the type. Here, the known AFPIII having the amino acid sequence closest to nfeAFP8 is an AFP isoform called HPLC12 purified from Macrozoarces americanus, which has been studied for many functions.

Example 4
Effect of adding a small amount (0.2 mM) of another activity-free AFP isoform (nfeAFP2) to the activity-free AFP isoform (nfeAFP6) alone, and an AFP isoform (nfeAFP8) also having activity in nfeAFP6 The effect of adding a small amount of (0.2 mM) was studied. The results are shown in FIG. Here, the numerical value on the horizontal axis in FIG. 5 indicates the concentration obtained by adding the molar concentrations of the two types of AFP isoforms.

  As shown in FIG. 5, the zero activity AFP isoform (nfeAFP6) showed strong thermal hysteresis activity by adding a small amount of active AFP isoform (+ nfeAFP8). On the other hand, even when an AFP isoform having zero activity was mixed (+ nfeAFP2), the thermal hysteresis activity remained zero. This experimental result shows that the AFP isoform belonging to the SP group which is not active alone exhibits high antifreeze activity when the AFP isoform belonging to the active QAE group coexists.

  AFP isoform nfeAFP6 belonging to the SP group which is not active alone was present in the most strongly absorbed peak numbered 2 in the HPLC elution pattern of FIG. This indicates that the AFP isoform of the SP group is most abundant in Nagagaji muscle. In Ocean pout (Macrozoares americanus), many SP groups AFP (11 types) are found, whereas only one type of QAE group is found. Analysis of amino acid sequence information suggests that the AFP isoform of the SP group has low hydrophobicity and high water solubility, and the water solubility of the AFP isoform of the SP group is actually higher than the AFP isoform of the QAE group It was. Therefore, the expression of the AFP isoforms of the SP group contributes to increasing the water solubility of the whole AFP isoform mixture, and this increases the concentration of AFP in fish serum to 20-30 mg / ml in the severe winter season. It is considered essential to increase

  From the above, it can be concluded that it is most effective to add AFP obtained from fish muscle to a hydrated product in the form of a mixture of positively charged AFP and negatively charged AFP. . By doing so, all types of AFP can be used effectively, and the presence of the SP group increases the solubility of the AFP mixture in water as a whole.

  The composition and method of the present invention include, for example, cereals, noodles, eggs, vegetables, fruits, meats, seafood, bread dough, ice confectionery, processed foods, medical products, diagnostic agents, reagents, cosmetics, lotions, blood, serum. Prevents the deterioration of taste caused by ice crystal growth or freeze-concentration and destruction of internal structure in the blood, etc., and is also promising in preservation solutions for platelets, sperm, ova, single cells, multicells, biological tissues, heart, pancreas, liver, kidney, etc. It is. Furthermore, it is also expected in a cold heat supply system or cold heat storage using a snow melting agent, a frost damage preventing agent, or ice slurry. The present invention greatly contributes to the promotion of efficient use of antifreeze proteins or the development of applied research on antifreeze proteins.

It is a table comparing old technology with recent technology regarding antifreeze proteins derived from fish. Upper panel: a diagram showing the elution pattern of antifreeze protein isoform derived from Nagagaji muscle by HPLC chromatography. Lower panel: Photographs of ice crystals observed for peaks 1 to 14 of HPLC. Bipyramidal ice crystals were observed for peaks 2-14. The second peak with the highest absorption was identified as nfeAFP6, which is an AFP isoform belonging to the SP group. It is the table | surface which classified and arranged each amino acid sequence about 13 types of AFP isoforms derived from Nagagaji into SP group and QAE group. It is the figure which plotted the protein concentration dependence of the thermal hysteresis activity (antifreeze activity) about five types of AFP isoforms in FIG. All measured values are thermal hysteresis activity per mole of AFP isoform monomer. The activity of the AFP isoform belonging to the SP type was zero. Upper: A plot showing that the AFP of the SP group (nfeAFP6), which has zero activity by itself, changes to have a high activity by adding a trace amount (0.2 mM) of AFP of the QAE group. Bottom: nfeAFP6 is a plot showing that activity does not change with the addition of AFP in the same SP group (0.2 mM). The numerical value on the horizontal axis indicates the concentration obtained by adding the molar concentrations of the two types of AFP isoforms.

Claims (11)

The following (a) and (b):
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 12,
(B) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 12, and having antifreeze activity
A positively charged antifreeze protein selected from (c) and (d) below:
(C) a protein comprising the amino acid sequence represented by SEQ ID NO: 16,
(D) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 16 and having antifreeze activity
The antifreeze protein is negatively charged chosen a composition comprising as an active ingredient from
The composition comprising a positively charged antifreeze protein and a negatively charged antifreeze protein in a molar ratio of 99: 1 to 80:20 . For lowering the freezing point of the hydrate, according to claim 1 Symbol placement of the composition. For inhibiting freeze concentration in water was, according to claim 1 Symbol placement of the composition. For inhibiting recrystallization of ice in water was claim 1 Symbol placement of the composition. For storing water-containing product, according to claim 1 Symbol placement of the composition. A method for lowering the freezing point of a hydrated material,
The following (a) and (b):
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 12,
(B) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 12, and having antifreeze activity
A positively charged antifreeze protein selected from (c) and (d) below:
(C) a protein comprising the amino acid sequence represented by SEQ ID NO: 16,
(D) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 16 and having antifreeze activity
Look including the addition of the antifreeze protein is negatively charged selected from, the antifreeze proteins are charged antifreeze protein and negatively positively charged 99: 1 to 80: 20 added in a molar ratio, said method. A method for inhibiting freeze concentration in a hydrated product, comprising:
The following (a) and (b):
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 12,
(B) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 12, and having antifreeze activity
A positively charged antifreeze protein selected from (c) and (d) below:
(C) a protein comprising the amino acid sequence represented by SEQ ID NO: 16,
(D) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 16 and having antifreeze activity
Look including the addition of the antifreeze protein is negatively charged selected from, the antifreeze proteins are charged antifreeze protein and negatively positively charged 99: 1 to 80: 20 added in a molar ratio, said method. A method for inhibiting recrystallization of ice in a water-containing material, the method comprising:
The following (a) and (b):
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 12,
(B) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 12, and having antifreeze activity
A positively charged antifreeze protein selected from (c) and (d) below:
(C) a protein comprising the amino acid sequence represented by SEQ ID NO: 16,
(D) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 16 and having antifreeze activity
Look including the addition of the antifreeze protein is negatively charged selected from, the antifreeze proteins are charged antifreeze protein and negatively positively charged 99: 1 to 80: 20 added in a molar ratio, said method. A method for preserving a hydrated product, comprising:
The following (a) and (b):
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 12,
(B) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 12, and having antifreeze activity
A positively charged antifreeze protein selected from (c) and (d) below:
(C) a protein comprising the amino acid sequence represented by SEQ ID NO: 16,
(D) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 16 and having antifreeze activity
Look including the addition of the antifreeze protein is negatively charged selected from, the antifreeze proteins are charged antifreeze protein and negatively positively charged 99: 1 to 80: 20 added in a molar ratio, said method. The following (a) and (b):
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 12,
(B) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 12, and having antifreeze activity
A positively charged antifreeze protein selected from (c) and (d) below:
(C) a protein comprising the amino acid sequence represented by SEQ ID NO: 16,
(D) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 16 and having antifreeze activity
A method of enhancing the antifreeze activity of a positively charged antifreeze protein by adding a negatively charged antifreeze protein selected from Adding negatively charged antifreeze protein such that the ratio of positively charged antifreeze protein to negatively charged antifreeze protein is a molar ratio of 99: 1 to 80:20. Item 11. The method according to Item 10.

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