JP4235083B2 - Antimicrobial proteins derived from microorganisms - Google Patents

Description

The present invention relates to a microorganism-derived antifreeze protein useful for uses such as food and drink, pharmaceuticals, and research material storage. That is, the present invention relates to a cryogenic microorganism or an antifreeze protein derived from a microorganism derived from the genus Flavobacterium which is a cryogenic microorganism.
The present invention also relates to a microorganism containing or producing an antifreeze protein having thermal hysteresis activity and a method for producing the antifreeze protein.
Furthermore, the present invention relates to a composition and method that utilize the effect of suppressing the growth of ice crystals and the effect of reducing the freezing point of an aqueous solution using an antifreeze protein derived from a microorganism.

  Antifreeze proteins are known to be produced by plants, fish, and insects. The antifreeze protein recognizes the ice crystal surface and adsorbs to the ice crystal surface to suppress the ice crystal growth. In other words, ice recrystallization during cryopreservation and thawing is prevented as much as possible. It is also a substance known to have an action of lowering the freezing temperature of water. The degree of activity of antifreeze protein is indicated by the thermal hysteresis activity as an index. Usually, the freezing temperature and the melting temperature of water are the same, but if antifreeze protein is present, it binds to ice crystals, so that the freezing temperature of water is lower than usual. The difference between the melting temperature of ice and the freezing temperature of water generated at this time is called “thermal hysteresis”, and the higher this value, the higher the activity of antifreeze protein. Antifreeze protein has been pointed out to be useful in controlling frost damage, improving the quality of frozen foods, preserving cells and tissues, and being used in cryosurgery. Addition to ice confectionery such as ice cream (see Patent Document 1) Liposome membrane stabilization (see Patent Document 2) is expected. Antifreeze protein is said to have the effect of improving the feel of the tongue produced from the growth of ice crystals that occur during freezing and suppressing the occurrence of drip after thawing. The anti-freeze protein thermal hysteresis activity is generally 0.2-0.5 ° C for plant-derived antifreeze protein and 0.7-1.5 ° C for fish-derived antifreeze protein, which is also not good for insects. It is known that there is frozen protein. In particular, antifreeze proteins derived from microorganisms are crude antifreeze proteins obtained after low-temperature acclimation at 3 ° C for Rhodococcus erythropolis and Micrococcus cryophilus. It is known to have thermal hysteresis activity of 35 ± 0.11 ° C. or 0.29 ± 0.01 ° C. (see Non-Patent Document 1). In addition, Pseudomonas putida GR12-2 is an antifreeze protein of a crude product obtained after acclimatization at a low temperature of 5 ° C. and is 0.11 ± 0.02 ° C. (see Non-Patent Document 2) It has been known. However, all of these have low thermal hysteresis activity, and higher activity is desired.

U.S. Pat. No. 4,725,500 US Pat. No. 5,869,092 CRYOBIOLOGY 30: 322-328 (1993) Can.J.Microbiol. 41: 776-784 (1995)

As described above, antifreeze proteins have been pointed out to be useful in various industrial fields, and many attempts have been made. However, known antifreeze proteins derived from plants, fish and microorganisms have low thermal hysteresis activity and have problems in safety and productivity, and have not yet been put into practical use.
The inventors of the present invention have obtained a novel antifreeze protein having high thermal hysteresis activity, a method for producing the antifreeze protein, a novel method for producing the antifreeze protein, which can be produced safely and inexpensively with high production efficiency. The purpose is to provide usage of antifreeze protein.

  In order to solve the above-mentioned problems that are extremely difficult to solve, the present inventors have conducted extensive research to provide an extremely effective and effective method, and as a result, a low-temperature microorganism containing the genus Flavobacterium, It was found that antimicrobial proteins derived from microorganisms with extremely high thermal hysteresis activity were produced.

The present invention relates to a microorganism-derived antifreeze protein having a thermal hysteresis activity of 0.5 ° C. or higher. The microorganism producing the antifreeze protein is, for example, a low-temperature microorganism including a microorganism belonging to the genus Flavobacterium.
The present invention relates to a method for producing an antifreeze protein derived from a microorganism having a very high thermal hysteresis activity using the microorganism. First, an antifreeze protein having extremely high thermal hysteresis activity is produced by culturing the microorganism at 20 ° C. or lower. For example, after the microorganism is induced or accumulated in a temperature environment of 20 ° C. or lower, 0 The present invention relates to a production method for obtaining a microorganism-derived antifreeze protein having a thermal hysteresis activity of 5 ° C. or higher.
The physicochemical property of the antifreeze protein derived from a microorganism having high thermal hysteresis activity according to the present invention is that the thermal hysteresis activity of a protein solution having an antifreeze protein concentration of 1.0 mg / ml or higher is 0.5 ° C. or higher. Thermal hysteresis activity increases in the presence of ammonium ions. Furthermore, even after heat treatment at 50 ° C. for 30 minutes in the presence of an aqueous solution of pH 8.0, it has heat resistance as a residual activity rate of 80% or more. The pH is stable in the range of 7-9. The molecular weight is about 19 kDa by sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis.
The present invention provides a polypeptide having thermal hysteresis activity and consisting of amino acids represented by SEQ ID NO: 1 and / or SEQ ID NO: 2. That is, the antifreeze protein derived from a microorganism having high thermal hysteresis activity of the present invention has a polypeptide sequence consisting of the amino acids represented by SEQ ID NO: 1 and / or SEQ ID NO: 2 in a part or all of the peptide sequence. The antifreeze protein derived from the microorganism of the present invention has a polypeptide sequence having 80% or more homology with the polypeptide sequence consisting of the amino acid represented by SEQ ID NO: 1 and / or SEQ ID NO: 2, and thermal hysteresis. A polypeptide having activity is included. Furthermore, the present invention provides a method for producing the polypeptide.
The present invention provides a polynucleotide sequence encoding a polypeptide having thermal hysteresis activity and consisting of the amino acids represented by SEQ ID NO: 1 and / or SEQ ID NO: 2. That is, the present invention relates to a polynucleotide encoding a polypeptide sequence consisting of the amino acid represented by SEQ ID NO: 1 and / or SEQ ID NO: 2 in part or all of the peptide sequence of an antifreeze protein derived from a microorganism. Further, a part or all of the peptide sequence of the antifreeze protein derived from the microorganism of the present invention has a polymorphism having 80% or more homology with the polypeptide sequence consisting of the amino acid represented by SEQ ID NO: 1 and / or SEQ ID NO: 2. The present invention relates to a polynucleotide encoding a peptide sequence and to a polynucleotide encoding a polypeptide having thermal hysteresis activity. Furthermore, the present invention provides a method for producing a microorganism-derived antifreeze protein using a polynucleotide encoding the polypeptide.

  The present invention relates to a composition containing an antifreeze protein derived from a microorganism having a thermal hysteresis activity of 0.5 ° C. or higher and a method of using the same. That is, the present invention is a composition using an antifreeze protein derived from a microorganism having an extremely high thermal hysteresis activity, and contains an antifreeze protein derived from a microorganism that can effectively exhibit the effect of inhibiting the growth of ice crystals and the effect of reducing the freezing point of an aqueous solution. The present invention relates to an ice crystal growth inhibitor that is a composition to be used, an ice crystal growth suppression method that is a method of using the effect, and a freezing point lowering method. For example, the method for using the ice crystal growth inhibitor containing an antifreeze protein derived from a microorganism uses an antifreeze protein derived from a microorganism having extremely high thermal hysteresis activity, for example, an organism such as an organ, tissue, cell, or microorganism, or The present invention can be widely used for a method for preserving biological materials in a low temperature region, and a method for modifying foods and beverages such as beverages, foods, and nutritional foods.

  The antifreeze protein derived from the microorganism of the present invention may be an antifreeze protein derived from a microorganism having a thermal hysteresis activity of 0.5 ° C. or higher, and if it is an antifreeze protein derived from the microorganism, it is an extract of the microorganism. Alternatively, the antifreeze protein derived from the microorganism isolated or purified may be used alone or as a mixture. The microorganism-derived antifreeze protein of the present invention may use at least a microorganism-derived antifreeze protein, and the microorganism strains to be produced may be used alone or in combination of two or more. The method for producing an antifreeze protein derived from a microorganism having a thermal hysteresis activity of 0.5 ° C. or higher according to the present invention may be performed in accordance with a normal method for extracting and purifying a protein from a living organism. The composition containing the antifreeze protein derived from the microorganism of the present invention may contain at least the antifreeze protein derived from the microorganism having the thermal hysteresis activity of 0.5 ° C. or higher of the present invention, and further enhances the thermal hysteresis activity. In addition, it can also be used in combination with substances including a potentiating substance, an antifreeze protein stabilizer of the present invention, a protective agent, and the like. In addition, the present invention also suppresses ice crystal growth, ice recrystallization inhibition, lowering of the freezing point of aqueous solution, inhibition of freezing (for example, frost damage prevention), stabilization of emulsification, microbial growth inhibition, discoloration prevention, and antioxidant prevention. The composition containing the antifreeze protein derived from microorganisms that is optimal for the above is included.

The microorganism of the present invention is not particularly limited as long as it is a microorganism producing an antifreeze protein derived from a microorganism having a thermal hysteresis activity of 0.5 ° C. or higher. For example, microorganisms that grow even in a low-temperature region of 20 ° C. or lower can be mentioned, and cryogenic microorganisms are particularly preferable. For example, the genera Flavobacterium, Pseudomonas, Micrococcus, Bacillus globis
porus), Candida sp., Candida scottii, Candida, Cladosporium herbarum, etc. And the like are preferred. Cytophaga genus is also preferred, but based on analysis such as gene level, Cytophaga genus is also reclassified as Flavobacterium genus (Int.J.Syst.Evol Microbiol. 50: 1055-1063 (2000)).

  The microorganism of the present invention is preferably a low-temperature microorganism belonging to the genus Flavobacterium, and the genus Flavobacterium of the present invention is an aerobic microorganism that decomposes and grows polysaccharides such as cellulose and chitin. is there. The genus Flavobacterium of the present invention includes Flavobacterium xanthum, Flavobacterium hydatis, Flavobacterium columnare, Flavobacterium flevense. ), Flavobacterium pectinovorum, Flavobacterium psychrophilum, Flavobacterium saccharophilum, Flavobacterium succinicans, Flavobacterium succinicans, Flavobacterium succinicans Flavobacterium uliginosum) and the like, preferably Flavobacterium xanthum. In addition, any genus Flavobacterium having the ability to produce a microorganism-derived antifreeze protein having a thermal hysteresis activity of 0.5 ° C. or higher can be used. For example, strains such as IFO 14958, IFO 14960, IFO 14905, IFO 14962, and IFO 14972 have been deposited with IFO and ATCC under the accession numbers, and are all available from publicly known distribution agencies and corporations. In the present invention, mutant strains of the above strains can also be used. Mutant strains can be obtained by treatment with radiation such as ultraviolet rays and X-rays or chemical mutation agents (NTG, etc.).

  The antifreeze protein derived from the microorganism of the present invention can be produced from the above-mentioned microorganism and has a thermal hysteresis activity of 0.5 ° C. or higher, preferably 1.0 ° C. or higher, more preferably 3.0 ° C. or higher. . Thermal hysteresis activity increases in the presence of ammonium ions. Further, even after heat treatment at 50 ° C. for 30 minutes in the presence of 20 mM Tris-HCl buffer (pH 8.0), it has heat resistance as a residual activity rate of 80% or more. The pH is stable in the range of 7-9. The molecular weight is about 19 kDa by sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis. For example, antifreeze protein derived from Flavobacterium xanthum has a thermal hysteresis activity of 2.0 to 5.0 ° C. at a concentration of 1.0 mg / ml, and is trisodium citrate dihydrate. Thermal hysteresis activity is enhanced. Furthermore, the stable temperature range is 50 ° C. or lower, and the stable pH range is pH 7-9.

The present invention includes a polypeptide having the above-described thermal hysteresis activity and comprising an amino acid represented by SEQ ID NO: 1 and / or SEQ ID NO: 2.
The antifreeze protein derived from a microorganism having high thermal hysteresis activity of the present invention has a polypeptide sequence consisting of the amino acid represented by SEQ ID NO: 1 and / or SEQ ID NO: 2 in a part or all of the peptide sequence. The polypeptide of the present invention is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and / or SEQ ID NO: 2, and one or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 and / or SEQ ID NO: 2 At least 80% or more when calculated using the amino acid sequence represented by SEQ ID NO: 1 or 2, and BLAST (Basic local alignment search tool), FASTA (Fast all), etc. In particular, a polypeptide having an amino acid sequence having a homology of 95% or more and having thermal hysteresis activity can be mentioned. The number of amino acids to be deleted, substituted or added is not particularly limited, but is a number that can be deleted, substituted or added by a known method such as site-directed mutagenesis, for example, 1 to several tens, preferably Is 1-5.

The present invention includes a polynucleotide sequence that encodes a polypeptide having the above-described thermal hysteresis activity and consisting of the amino acid represented by SEQ ID NO: 1 and / or SEQ ID NO: 2.
The antifreeze protein derived from a microorganism having a high thermal hysteresis activity of the present invention is not particularly limited as long as it includes a polypeptide sequence consisting of the amino acid represented by SEQ ID NO: 1 and / or SEQ ID NO: 2 in part or all of the peptide sequence. It is not limited. The polynucleotide of the present invention includes both DNA and RNA. A polynucleotide encoding the polypeptide of the present invention, for example, a DNA consisting of a base sequence encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1, generally has a plurality of genetic codes for one amino acid. It exists, but is included in the DNA of the present invention if it encodes the polypeptide of the present invention. Furthermore, it has a base sequence having a homology of 80% or more with a base sequence that hybridizes under stringent conditions with a DNA having a base sequence complementary to the base sequence of the DNA of the present invention, and has thermal hysteresis activity. Also included is DNA encoding a polypeptide. Specifically, the hybridizable DNA encodes a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 when calculated using BLAST (Basic local alignment search tool), FASTA (Fast all), or the like. Examples thereof include DNA having at least 80% homology with the base sequence, preferably DNA having 95% or more homology. The polynucleotide according to the present invention may be derived from nature or may be totally synthesized, for example. Furthermore, you may synthesize | combine using a part derived from nature. A typical method for obtaining a polynucleotide according to the present invention is, for example, a method commonly used in the field of genetic engineering, for example, screening using an appropriate DNA probe prepared based on partial amino acid sequence information. The method etc. can be mentioned.
Furthermore, this invention relates to the manufacturing method of the antifreeze protein which has the thermal hysteresis activity which has the polypeptide of this invention.

  The production method for obtaining an antifreeze protein derived from a microorganism having a thermal hysteresis activity of 0.5 ° C. or higher according to the present invention is the antifreeze derived from a microorganism having a high thermal hysteresis activity as a result of the above-mentioned microorganism growing well. It is desirable to produce the protein under conditions that allow it to be produced. Depending on the nature of the microorganism, the pH of the medium, the culture period, etc. may be appropriately changed. For example, the culture method may be either liquid culture or solid culture, and preferably liquid culture is used. For example, the liquid culture can be performed as follows. As the medium that can be used, any medium can be used as long as it can grow microorganisms that produce antifreeze proteins derived from microorganisms having high thermal hysteresis activity. For example, carbon sources such as glucose, sucrose, glycerin, dextrin, molasses, organic acids, ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate, or peptone, yeast extract, corn steep liquor, casein hydrolyzate, meat Nitrogen sources such as extracts and further added with inorganic salts such as potassium salts, magnesium salts, sodium salts, phosphate salts, manganese salts, iron salts, zinc salts and the like can be used. The pH of the medium is adjusted to, for example, about 3 to 10, preferably about 7 to 8, and the culture temperature is usually about -20 to 20 ° C, preferably about 10 ° C or less, more preferably 7 ° C or less, and 1 to 20 The culture is carried out under aerobic conditions for a day, preferably about 3 to 15 days. As the culture method, for example, stationary culture, shaking culture, and aerobic culture using a jar fermenter can be used. The antifreeze protein derived from the microorganism of the present invention is produced and accumulated in the culture solution obtained by culturing the above microorganism of the present invention and / or the cultured microorganism. Furthermore, the antifreeze protein derived from the microorganism of the present invention can be obtained from the culture solution and / or the cultured microorganism by utilizing a normal extraction method and purification method. For example, when the antifreeze protein derived from the microorganism of the present invention accumulates in the cultured microorganism, the microorganism is cultured in a low temperature region, and then the culture solution is centrifuged to obtain the cultured microorganism. Subsequently, the cultured microorganisms are crushed by an appropriate method, and an extract containing antifreeze proteins derived from microorganisms having high thermal hysteresis activity can be obtained from the crushed liquid by centrifugation or the like. The extract may be used as it is as the microorganism-derived antifreeze protein of the present invention, but the microorganism-derived antifreeze protein is further isolated and purified, for example, centrifugation, UF concentration, salting out, dialysis, gel filtration chromatography. The purified product of antifreeze protein derived from a microorganism with high thermal hysteresis according to the present invention can be obtained by combining various types of chromatography such as chromatography and ion exchange resin and processing by a conventional method.

  The thermal hysteresis measurement of the antifreeze protein derived from the microorganism of the present invention is based on the principle of measuring the difference between the melting temperature and the freezing temperature of the aqueous solution. DeVries et al. (Methods Enzymol. 127, 293-303 (1986)). What is necessary is just to carry out according to the method. That is, it is performed by gradually cooling an aqueous solution containing the antifreeze protein derived from the microorganism and monitoring the freezing point of the aqueous solution. The freezing point is an uncontrollable crystal growth or a negligible temperature at which crystal growth occurs. The melting point is the highest temperature at which one ice crystal remains stable without melting.

  The ice crystal growth inhibitor and method for inhibiting ice crystal growth containing an antifreeze protein derived from a microorganism having a thermal hysteresis activity of 0.5 ° C. or higher according to the present invention are derived from a microorganism having a thermal hysteresis activity of 0.5 ° C. or higher. By containing at least antifreeze protein, it has the effect of inhibiting or / and inhibiting the growth of ice crystals as a solvent, and it can be used for frost control agents, cells / tissues of animals / plants, food preservation, etc. . The ice crystal growth inhibitor of the present invention can be formulated as a solution or powder form of the antifreeze protein derived from the microorganism, or in the form of a solution or paste according to the properties or processes of the product to be added. It can be processed into a dosage form that can be expected to be most effective. The ice crystal growth inhibitor of the present invention may be a mixed form of the antimicrobial protein derived from the microorganism of the present invention and other components. As such a component, one that does not impair the above-described effects of the antifreeze protein derived from the microorganism can be used. Furthermore, the ice crystal growth inhibitor containing the microorganism-derived antifreeze protein of the present invention can be used by appropriately selecting the microorganism-derived antifreeze protein under conditions such as optimum concentration and pH. .

  Examples of methods for reforming foods using microorganism-derived antifreeze proteins include fruits, seafood, noodles, gratin, cooked rice such as risotto, hamburger, bread, pizza, sponge cake, castella, hot cake, Frozen foods such as grilled, takoyaki, manju, dumpling, tsukuyane, soup, stew, sauce, tofu, bouillon, seasoning, cooked food, etc., for example, ice cream and Monaca When manufacturing frozen confectionery including skin, etc., the antifreeze protein derived from the microorganism of the present invention can be used as it is or in combination with other foods or food ingredients and used according to conventional methods. it can. The food according to the present invention may be in the form of any food or drink in the form of a solid (powder, granule, etc.), paste, liquid or suspension. By using the antifreeze protein of the present invention, even when the frozen food is stored frozen for a long period of time, a good texture and deliciousness can be maintained.

  The reducing agent and method for reducing the freezing point of an aqueous solution containing an antifreeze protein derived from a microorganism having a thermal hysteresis activity of 0.5 ° C. or higher according to the present invention is not derived from a microorganism having a thermal hysteresis activity of at least 0.5 ° C. or higher. By containing frozen protein, it has the action of lowering the freezing point of the solvent, and it can be used for preservation of frost damage control agents, cells / tissues such as animals and plants, and foods. The agent for reducing the freezing point of the aqueous solution of the present invention can be formulated into a solution or a powder form of the antifreeze protein derived from the microorganism, or in the form of a solution or paste according to the properties or processes of the product to be added. It can be processed into a dosage form that can be expected to be most effective. The agent for reducing the freezing point of the aqueous solution of the present invention may be a mixed form of the antifreeze protein derived from the microorganism of the present invention and other components. As such a component, one that does not impair the above-described effect of the antifreeze protein derived from microorganisms can be used. Furthermore, the agent for reducing the freezing point of an aqueous solution containing an antifreeze protein derived from a microorganism of the present invention can be used by appropriately selecting the antifreeze protein derived from the microorganism under conditions such as an optimal concentration and pH. it can.

  The method for reducing the freezing point of an aqueous solution containing an antifreeze protein derived from a microorganism having a thermal hysteresis activity of 0.5 ° C. or higher according to the present invention includes, for example, cells and tissues such as microorganisms, animals and plants, proteins such as blood and enzymes, The present invention relates to a method for preserving an organism that is a biological sample (biological material) such as a vaccine or a cellular extract. Furthermore, the antifreeze protein derived from the microorganism of the present invention is added to an aqueous solution containing organisms and stored, thereby reducing the freezing point of the aqueous solution and maintaining the viability and functionality of the biological sample. Has the effect of improving the period. For example, the method for preserving an organism of the present invention includes use during transplantation surgery or transport to a hospital. The microorganism-derived antifreeze protein of the present invention not only lowers the freezing point of an aqueous solution, but also has an effect of suppressing the growth of ice crystals that occur during storage below the freezing point. Utilizing this effect, it minimizes the degradation and denaturation of organisms and suppresses cell damage resulting from the growth of ice crystals. Moreover, it is thought that the antifreeze protein derived from the microorganism of the present invention contributes to the stabilization of the cell membrane, and improves the storage stability of the cells. Such cells to be preserved include bacteria, fungi including yeast, and particularly higher eukaryotic cells (eg, hybridoma strains or tissue culture cell lines). Cellular extracts include in vitro protein synthesis systems, enzyme preparations, samples containing membrane components, and the like.

  According to the present invention, a microorganism-derived antifreeze protein having a thermal hysteresis activity of 0.5 ° C. or higher can be provided. The antifreeze protein derived from microorganisms has an extremely high thermal hysteresis activity compared to the antifreeze proteins derived from plants, fish and microorganisms known so far, and therefore various anti-freeze proteins such as foods, pharmaceuticals, and research reagents can be used. It can be used in various industrial fields. Furthermore, by the method for producing a microorganism-derived antifreeze protein of the present invention, it has become possible to obtain a novel microorganism-derived antifreeze protein having excellent safety and productivity, high production efficiency, and the like.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited by a following example. In the examples of the present invention, thermal hysteresis measurement was applied when comparing the strength of antifreeze activity.

Cultivation of Flavobacterium xanthum IFO 14972 30 ml of TSB medium (manufactured by DIFCO) (3.0%) was dispensed into each 200 ml Erlenmeyer flask, sterilized at 121 ° C. for 20 minutes, cooled, Flavobacterium xanthum IFO 14972 was inoculated one platinum ear at a time and cultured with shaking at 5 ° C. for 1 week to prepare a seed culture solution. 3 L of the above TSB medium is put into a 5 L culture jar, sterilized at 121 ° C. for 20 minutes, cooled, then inoculated with 27 ml of the above seed culture solution, and aerated in the culture solution at 5 ° C. for 6 days. Stirring culture was performed. After completion of the culture, the cells were collected by centrifugation.

Purification of extract having thermal hysteresis activity from bacterial cells An extract of a microorganism-derived antifreeze protein having thermal hysteresis activity was obtained by the following steps.
1) Preparation of cell-free extract:
The cells obtained in the above culture are suspended in Tris / HCl buffer (20 mM, pH 8) and then cooled in an ice bath for 40 minutes with an ultrasonic crusher “INSONATOR 201M” (manufactured by Kubota Corporation). did. The disrupted solution was centrifuged to remove the cell residue, and a cell-free extract was obtained.

2) Purification by ammonium sulfate fraction:
Ammonium sulfate was added to the cell-free extract so that the saturation amount was 20% (w / v), and the mixture was gently stirred at 4 ° C. for 15 hours. The supernatant was collected by centrifugation at 10,000 g for 20 minutes at 4 ° C. Ammonium sulfate was added to the supernatant to a saturation amount of 60% (w / v), and the mixture was gently stirred at 4 ° C. for 7 hours. The precipitate was collected by centrifugation at 4 ° C., 10,000 g for 20 minutes, dissolved in Tris-HCl buffer (20 mM, pH 8), and collected as an extract. This extract was put into a dialysis tube (fraction molecular weight: 3,500) manufactured by Spectrum, and 66 hours were obtained by exchanging 5 L of Tris / HCl buffer solution (20 mM, pH 8) used as an external solution for dialysis three times. Dialysis was performed to obtain an extract.

3) Purification by ion exchange chromatography:
The extract was passed through a glass column (1.0 cm in diameter x 5.8 cm in height) packed with DEAE-Cellulofine previously equilibrated with Tris-HCl buffer (20 mM, pH 8) and washed with the same buffer. The active fraction was collected. This active fraction was concentrated with a freeze dryer “EYELA FREEZE DRYER FD-1” (manufactured by Tokyo Rika Kikai Co., Ltd.) and dissolved in distilled water. The extract obtained here had a protein concentration of 1 mg / ml and a thermal hysteresis activity of 2.5 to 4.7 ° C. The thermal hysteresis activity was measured by the method described in Example 3 below.

4) Purification by hydrophobic chromatography:
A glass column (diameter 0.8 cm × diameter) packed with Butyl-Toyopearl, which was added with ammonium sulfate to a concentration of 20% and equilibrated with Tris-HCl buffer (20 mM, pH 7.5) containing 20% ammonium sulfate in advance. The solution was passed through a height of 5.4 cm), washed with the same buffer solution, and then the active fraction was collected by a linear concentration gradient to a tris-HCl buffer solution (20 mM, pH 7.5) containing no ammonium sulfate. Further, this active fraction was dialyzed with Tris-HCl buffer (5 mM, pH 8), added with ammonium sulfate to a concentration of 20%, and equilibrated in advance with Tris-HCl buffer (20 mM, pH 7.5) containing 20% ammonium sulfate. The solution was passed through a glass column filled with Butyl-Toyopearl (directly 0.8 cm × height 5.4 cm), washed with the same buffer solution, and then added to a tris-HCl buffer solution (20 mM, pH 7.5) containing no ammonium sulfate. Active fractions were collected in a linear concentration gradient. The active fraction was dialyzed against Tris-HCl buffer (5 mM, pH 8) and concentrated.

5) Purification by gel filtration chromatography:
The concentrated solution was dissolved in Tris-HCl buffer (50 mM, pH 7.5) containing 0.2 M NaCl, and gel filtration chromatography TSKgel G3000SW (column size: diameter 0.75 cm × diameter previously equilibrated with the same buffer). Elution with the same buffer solution. The obtained active fraction was concentrated, dialyzed against Tris-HCl buffer (5 mM, pH 8), and concentrated to obtain an antifreeze protein solution.

Measurement of thermal hysteresis activity The thermal hysteresis of the aqueous solution containing the antifreeze protein derived from the microorganism of the present invention is measured by gradually cooling the aqueous solution containing the antifreeze protein derived from the microorganism and monitoring the freezing point of the aqueous solution. Is done. The freezing point is an uncontrollable crystal growth or a negligible temperature at which crystal growth occurs. The melting point is the highest temperature at which one ice crystal remains stable without melting. In the present Example, it carried out according to the method of Devries et al. (Methods Enzymol. 127, 293-303 (1986)). Specifically, thermal hysteresis was measured by the following method. A sample of 1 μl was placed on a circular glass having a diameter of 16 mm and a thickness of 0.12 mm. The slide was placed on a temperature-controlled microscope stage (LK-600PM manufactured by Linkham Co., Ltd.), and the stage was rapidly frozen to −40 ° C. at a rate of 50 ° C./min in order to make a lot of fine ice. While monitoring ice crystals with a microscope, the stage temperature was gradually increased at a rate of 0.02 ° C./min, and the temperature at which the last crystal disappeared was defined as the melting point. Again, the stage was snap frozen to −40 ° C. at a rate of 50 ° C./min to make a lot of fine ice. The stage temperature was gradually increased at a rate of 0.02 ° C./min to melt ice, and reached an equilibrium temperature at which one ice crystal nuclide having a diameter of about 10 μm was formed. Thereafter, the temperature was decreased at a rate of 1 ° C./min, and the size of ice crystals was monitored. In samples that do not contain antifreeze proteins, ice crystals begin to grow rapidly, so the melting and freezing temperatures are the same. On the other hand, when antifreeze protein is present, the size of the ice crystal remains constant for a while when the temperature drops, and the ice crystal grows dramatically at the hysteresis freezing point. That is, a difference occurs between the melting temperature and the freezing temperature. This temperature difference of thermal hysteresis depends on the concentration and specific activity of antifreeze protein. In this invention, the average value of the value measured 3 times was employ | adopted as a thermal hysteresis value.
The protein concentration was measured using a protein concentration measurement kit manufactured by BIO RAD Co., Ltd. In the measurement, a 0.2 mg / ml BSA solution was used as a standard solution, and the average value of three measurements was used for all.

Characterization of extracts with thermal hysteresis activity from bacterial cells
The purified antifreeze protein solution obtained in 5) of Example 2 was examined for temperature stability, pH stability, molecular weight, and internal amino acid sequence. In addition, the effects of addition of organic acids, sugars and salts on thermal hysteresis activity were investigated, and the effect of inhibiting ice recrystallization was investigated. In addition, the measurement of the thermal hysteresis activity of the antifreeze protein derived from microorganisms was also performed according to the method shown in Example 3.

1. Temperature stability:
The temperature stability of the purified antifreeze protein solution obtained in 5) of Example 2 was measured.
50 μl of a purified solution (protein concentration: 10 mg / ml) containing antifreeze protein derived from microorganisms is placed in a 1.5 ml Eppendorf tube, and then kept at 40, 50, 60, and 70 ° C. for 30 minutes. After maintaining at each temperature, the sample was immediately cooled on ice, and the thermal hysteresis activity of the antifreeze protein derived from the microorganism was measured. In addition, the ratio is calculated from the activity value at each temperature when the thermal hysteresis activity of the antifreeze protein derived from microorganisms kept at 0 ° C. for 30 minutes without heat treatment is 100%, and is shown as the residual activity rate. The temperature stability was evaluated.
As a result, regarding the temperature stability of the antifreeze protein derived from the microorganism, the residual activity from 0 to 50 ° C. was about 90% or more, and there was almost no residual activity at 60 ° C. Therefore, it was suggested that the antifreeze protein derived from the microorganism of the present invention is stable up to a temperature of 0 ° C. to 50 ° C. and is an antifreeze protein derived from a microorganism capable of effectively maintaining thermal hysteresis activity.

2. pH stability:
The pH stability of the purified antifreeze protein solution obtained in 5) of Example 2 was measured.
Extract containing microorganism-derived antifreeze protein (protein concentration: 10 mg / ml), acetic acid buffer (pH 4.1 or pH 5.0), potassium phosphate buffer (pH 7.2), or glycine-NaOH buffer Five types of buffer solutions (pH 8.9 or 9.9) were used, and each buffer solution was added to a final concentration of 20 mM. Solutions containing antifreeze proteins derived from microorganisms in each pH environment were stored at 4 ° C. for 11 days, and thermal hysteresis activity was measured over time, 0 hours, 1 day, 3 days, 7 days, and 11 days later.
As a result, it was suggested that the antifreeze protein derived from the microorganism is stable at pH 7-9. Therefore, it was suggested that the microorganism-derived antifreeze protein in this pH range is a microorganism-derived antifreeze protein that is stable in the vicinity of neutrality and can effectively maintain thermal hysteresis activity.

3. Molecular weight (gel filtration method):
The molecular weight of the purified antifreeze protein solution after gel filtration obtained in 5) of Example 2 was measured by gel filtration chromatography. Here, TSKgel G3000SW (column size: diameter 0.75 cm × length 60 cm) was used as a packing material, and Tris-HCl buffer solution (50 mM, pH 7.5) containing 0.2 M NaCl was used as an eluent. As standard proteins, vitamin B12 [molecular weight 1,350], horse myoglobin [molecular weight 17,000], chicken egg white albumin [molecular weight 44,000], bovine gamma globulin [molecular weight 158,000], thioglobulin [molecular weight 670,000] A calibration curve was prepared using the protein. As a result, it was found that the antifreeze protein solution obtained in 5) of Example 2 was purified to single. By this method, the molecular weight of the antifreeze protein according to the present invention was determined to be about 38 kDa.

4. Molecular weight (SDS polyacrylamide electrophoresis):
The molecular weight of the purified antifreeze protein solution after gel filtration obtained in 5) of Example 2 was measured by SDS polyacrylamide gel electrophoresis. Electrophoresis was performed using a separation gel “Pagel SPG-520L” (manufactured by ATTO). As a buffer in the gel, 25 mM trishydroxymethylaminomethane and 192 mM glycine (pH 8.6) containing 0.1% SDS were used. After electrophoresis at 20 mA for 1.2 hours, the protein was stained with CBB R-250. A single band was confirmed at a position of about 19 kDa. Combined with the above results, the antifreeze protein according to the present invention was found to be a dimer of the same subunit.

5. Internal amino acid sequence:
The purified antifreeze protein solution obtained after gel filtration obtained in 5) of Example 2 was fragmented by cyanogen bromide decomposition, and each peptide fragment was fractionated by SDS polyacrylamide electrophoresis. Two of the peptide fragments were subjected to a protein sequencer and sequenced. The determined partial amino acid sequence is shown below. Note that (N) indicates an N terminal, and (C) indicates a C terminal.

SEQ ID NO: 1;
(N) −Leu Glu Ala Gln Lys Gln Leu Asp Lys Leu Ser Thr Thr Tyr Asp Ala Asp − (C)

SEQ ID NO: 2;
(N) −Glu Glu Tyr Gln Gly Lys Lys Tyr Glu Ala Glu Ala Ala Thr Val Thr Glu Ala Val Asn Gly Glu − (C)

6. Effects of adding organic acids, sugars and salts:
With respect to the purified antifreeze protein solution after gel filtration obtained in 5) of Example 2, the effect of enhancing thermal hysteresis activity was measured.
To a purified solution (protein concentration: 10 mg / ml) containing antifreeze protein derived from microorganisms, trisodium citrate dihydrate, monosodium succinate, sodium acetate monohydrate, lactic acid, ammonium sulfate, sorbitol, sucrose, Sodium L-glutamate monohydrate and sodium L-aspartate were added with each additive adjusted to a final concentration of 100 mM, 500 mM or 1 M, respectively. The thermal hysteresis activity under each condition was measured. In addition, distilled water was added to the antifreeze protein derived from microorganisms and used as a control.
As a result, an effect of enhancing the thermal hysteresis activity of the antifreeze protein derived from the microorganism by the additive was recognized. The enhancing effect varies depending on each additive, and the additive having a higher effect on the thermal hysteresis activity of the antifreeze protein derived from the microorganism of this example is 500 mM trisodium citrate dihydrate, 500 mM monosodium succinate, 500 mM sodium L-glutamate monohydrate, 1M sorbitol.

7. Effect of inhibiting ice recrystallization:
The purified antifreeze protein solution obtained after the gel filtration obtained in 5) of Example 2 was evaluated for the effect of inhibiting recrystallization of ice.
An antifreeze protein sample containing water was prepared to a sucrose concentration of 30%, and 1 μl of the sample was placed on a round glass slide having a diameter of 16 mm. A glass slide having the same shape was placed thereon, and a 200 g weight was applied to the sample in order to make the thickness of the slide uniform. The sample was placed on a Linkam L-600A temperature controlled microscope stage, and the stage was snap frozen at a rate of 50 ° C / min to minus 40 ° C in order to make a lot of small ice. The stage temperature was rapidly raised to minus 9 ° C., the temperature was kept constant, and the ice size was observed with a microscope. Ice crystals were recorded after 0, 10 and 30 minutes with a microscope camera. The diameters of the ice crystals started from 5 μm or less after 0 minutes. However, when water was used instead of the antifreeze protein sample as a control, the ice crystals recrystallized after 10 to 30 minutes and the average diameter was 5 μm. It grew to reach to 10 μm. On the other hand, when a sucrose solution containing 1 mg / ml of antifreeze protein sample was used, the average diameter of ice crystals was 5 μm or less even after 30 minutes.

  From the results of the above examples, it was suggested that the antifreeze protein according to the present invention inhibits ice crystal growth by inhibiting ice recrystallization. Therefore, cell death caused by freeze concentration and ice crystal growth caused by cryopreservation of cells and tissues, etc., and in foods, taste, crunchiness, deterioration of freshness, or nutritional components such as vitamin C and protein It is expected that various problems such as decomposition of useful components such as chlorophyll can be avoided.

Claims (9)

Antifreeze proteins derived from microorganisms belonging to the genus Flavobacterium having the following physicochemical properties:
(1) Thermal hysteresis activity: 0.5 ° C. or higher,
(2) After the heat treatment at 50 ° C. for 30 minutes in the presence of an aqueous solution of pH 8.0, the residual activity rate is 80% or more,
(3) Stable pH range: pH is stable in the range of 7-9,
(4) Molecular weight: about 19 kDa by sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis. The antifreeze protein derived from a microorganism according to claim 1, wherein the microorganism belonging to the genus Flavobacterium is Flavobacterium xanthum. SEQ ID NO: 1 and / or SEQ ID NO microbial antifreeze protein of claim 1 comprising a polypeptide consisting of the amino acid sequence represented by 2. A method of producing antifreeze proteins derived from microorganisms, a 10 ° C. or less, according to claim 1 antifreeze process for producing a protein derived from a microorganism according made using the process of culturing at pH 3-10. An ice crystal growth inhibitor comprising the microorganism-derived antifreeze protein according to claim 1 . A method for inhibiting ice crystal growth, comprising using the microorganism-derived antifreeze protein according to claim 1 . A method for lowering the freezing point of an aqueous solution using the antifreeze protein derived from a microorganism according to claim 1 . A method for preserving an organism using the microorganism-derived antifreeze protein according to claim 1 . A method for modifying a food comprising the antimicrobial protein derived from microorganisms according to claim 1 .