JP2011231089A - Antifreeze substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an antifreeze substance that can efficiently be produced at a low cost on an industrial scale, has a sufficiently high antifreeze activity, is safe, and has antifreeze activity excellent to the extent usable in practical applications, and to provide a polypeptide, i.e. an active segment of the antifreeze substance, and an antibody, a composition, a foodstuff, an agent for protecting an organism specimen, and cosmetics, each comprising the antifreeze substance or the polypeptide.SOLUTION: The antifreeze substance is derived from a plant and includes at least one specific protein, which plant is selected from the group consisting of plants belonging to Brassicaceae, Umbelliferae, Liliaceae and Compositae, and an analogue or an improved variety thereof.

Description

  The present invention relates to an ice crystallization inhibitor, a polypeptide which is an active part thereof, and an antibody, a composition, a food, a biological sample protective agent and a cosmetic containing the ice crystallization inhibitor or polypeptide.

  Ice crystallization inhibitor is a substance also called antifreeze protein or AFP, and it is a protein that has the function of adsorbing on the surface of ice crystals and hindering its growth. In living organisms, it is used to protect itself from freezing of cells. Has been. Such an ice crystallization inhibitor is found from, for example, fish, insects, plants, fungi, microorganisms, etc. (Patent Documents 1 and 2, Non-Patent Documents 1 to 4).

  However, the ice crystallization inhibitors reported so far are present in living organisms such as fish, plants, insects, fungi and bacteria, and the extraction efficiency is very poor or even if they are present in large quantities. However, it has a problem that it is difficult to capture and cultivate the organism itself, and it cannot be industrially produced and used for food.

  Therefore, as a method for providing an ice crystallization inhibitor having antifreeze activity stably and inexpensively throughout the year, a method of extracting daikon radish stored at low temperature with water or the like has been proposed (Patent Document 3). However, the ice crystallization inhibitory substance obtained by the method described in Patent Document 3 does not necessarily have sufficient ice crystallization inhibitory activity.

JP 2004-24237 A JP 2004-275008 A JP 2007-153834 A

Biophysics, Vol. 43, No. 3, pp. 130-135 (2003) Plant Physiology, Volume 119, Pages 1361 to 1369 (1999) Biochem. J. (Biochemical Journal), 340, 385-391 (1999) Can. J. Microbiol. (Canadian Journal of Microbiology), vol. 144, p. 6 (1998)

  An object of the present invention is to provide an ice crystallization inhibitor that can be produced efficiently and inexpensively at an industrial level, has a sufficiently high ice crystallization inhibitory activity, and has an excellent ice crystallization inhibitory activity that is safe and practical. Is to provide. The present invention also provides a polypeptide which is an active part of the ice crystallization inhibitor, and an antibody, composition, food, biological sample protecting agent and cosmetic containing the ice crystallization inhibitor or polypeptide. Objective.

  The present inventors have intensively studied to solve the above problems. As a result, for example, it has been found that a protein having a specific molecular weight obtained from silkworm radish has a high ice crystallization inhibitory effect, and the present invention has been completed.

The ice crystallization inhibitor according to the present invention is derived from a plant and contains at least one protein of any one of (1) to (4) below.
(1) Protein having the amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 1) and having a molecular weight of 19,000 ± 200 Da measured by SDS-PAGE (2) SEQ ID NO: 2 (SEQ ID NO: 2) (3) The molecular weight measured by SDS-PAGE is 59,000 ± 600 Da, and its N-terminal is modified. Receiving protein (4) A protein in which one or more amino acids of the amino acid sequence of any one of (1) to (3) above are deleted, substituted or added, and has ice crystallization inhibitory activity protein.

  The plant containing the ice crystallization inhibitor according to the present invention includes one or more plants selected from the group consisting of Brassicaceae, Apiaceae, Lilyaceae and Asteraceae, or their related varieties or improved varieties. . The plant belonging to the Brassicaceae family is at least one plant selected from the group consisting of Chinese cabbage, Japanese radish, broccoli, Chingensai, Komatsuna, turnip, Sirona, Nozawana, Hiroshima rape, Mizuna and mustard, or their related varieties. Or an improved variety can be mentioned. Furthermore, examples of the plant belonging to the Brassicaceae family include radish (Raphanus sativus) or related varieties or improved varieties thereof, and radish sprout that is a sprout of radish.

  More specifically, examples of the ice crystallization inhibitor according to the present invention include those contained in a fraction that precipitates at an acetone concentration of 30% by volume in an acetone fraction.

  The polypeptide according to the present invention is obtained by dissociating the above-mentioned ice crystallization inhibitor according to the present invention, and has an ice crystallization inhibitory activity.

  The antibody, the composition, the food, the biological sample protective agent and the cosmetic according to the present invention are characterized by including the ice crystallization inhibitor and / or the polypeptide according to the present invention.

  The ice crystallization inhibitory substance according to the present invention can be obtained in a large amount at a low cost from, for example, food radish, which is a food, and has high ice crystallization inhibitory activity. Further, by adding the ice crystallization inhibitor of the present invention to food, it can be used for maintaining the quality of frozen food. Furthermore, the ice crystallization inhibitor of the present invention can also be used effectively for cryoprotectants and cosmetics (skin protectants) in the cryopreservation of organs, cells and blood (platelets).

  The ice crystallization inhibitor according to the present invention is derived from a plant and contains at least one protein of molecular weight 19,000 ± 200 Da, 22,000 ± 250 Da or 59,000 ± 600 Da by SDS-PAGE. .

  In the present invention, the ice crystallization inhibiting substance widely means a substance having a function of inhibiting the growth of ice crystals, and is well-known such as measurement of thermal hysteresis, observation of ice crystal structure, measurement of ice crystallization inhibitory activity. As long as ice crystallization inhibitory activity is exhibited by any of the methods, it is included in the present invention. Thermal hysteresis refers to the temperature range in which ice cannot grow in an aqueous solution of an ice crystallization inhibitor substance even at a temperature below the equilibrium melting point.If the temperature at which ice begins to grow in an aqueous solution is defined as the freezing point, thermal hysteresis is defined. Is detected as the difference between the equilibrium melting point and the freezing point.

  The ice crystallization inhibitor according to the present invention can be obtained from plants. Although it does not specifically limit as a plant, For example, the plant which belongs to the Brassicaceae, the Aceraceae, the Lilyaceae, or the Asteraceae is mentioned. Plants belonging to the Brassicaceae include Chinese cabbage, Japanese radish, broccoli, Chingensai, Komatsuna, turnip, Sirona, Nozawana, Hiroshima rape, Mizuna, mustard and the like. Examples of the plant belonging to the family Aceraceae include carrots, examples of the plant belonging to the family Liliaceae include leeks, and examples of the plant belonging to the family Asteraceae include spring chrysanthemums. Similar varieties and improved varieties of these plants can also be used as appropriate. The ice crystallization inhibitor according to the present invention is not particularly limited, but can be suitably obtained from, for example, radish seeds. Although it does not specifically limit as a radish seed | species, For example, a radish, a Japanese radish, and a radish can be illustrated. Moreover, if the sprout of the said plant, especially the radish sprout which is a sprout of a radish, the ice crystallization inhibitory substance which concerns on this invention can be obtained efficiently.

  You may use in the state which induced | guided | derived the ice crystallization inhibitory substance in a plant by well-known methods, such as acclimatizing these plants at low temperature. The temperature for low temperature acclimation is not particularly limited, but the lower limit temperature is preferably 0 ° C. or higher, and the upper limit temperature is preferably 20 ° C. or lower. The period of low temperature acclimation is not particularly limited, but it is preferably 3 days or more.

  Hereinafter, the properties of the ice crystallization inhibitor according to the present invention will be described in detail. The ice crystallization inhibitor includes at least one protein having a molecular weight of 19,000 ± 200 Da, 22,000 ± 250 Da, or 59,000 ± 600 Da by SDS-PAGE.

  A protein having a molecular weight of 19,000 ± 200 Da has an amino acid sequence substantially homologous to the amino acid sequence of SEQ ID NO: 1, and a protein having a molecular weight of 22,000 ± 250 Da has an amino acid sequence substantially homologous to SEQ ID NO: 2. However, the 59,000 ± 600 Da protein has some N-terminal modification. Here, the amino acid sequences being substantially homologous means that at least 80% or more, more preferably 90% or more, and most preferably 95 to 100% of the amino acid sequences overlap.

  Further, the ice crystallization inhibitor according to the present invention is a protein in which one or a plurality of amino acids in the amino acid sequence of any one of the above proteins is deleted, substituted or added, and has an ice crystallization inhibitory activity. May be included. In the above amino acid sequence, the number of amino acids to be deleted, substituted or added is preferably 1 or more, 3 or less, more preferably 1 or 2, and particularly preferably 1.

  Having an ice crystallization inhibitory activity means having an activity to inhibit ice crystal growth and coarsening by inhibiting ice crystallization or controlling the ice crystal shape in the protein solution. To do.

  The ice crystallization inhibitor of the present invention is obtained as an adsorbed fraction by chromatography using an anion exchange resin under the condition of pH 8.0. Moreover, it precipitates suitably at an acetone concentration of 30% by volume or less. Here, the anion exchanger is not particularly limited, and examples thereof include DEAE (Diethylaminoethyl) and Q (Quaternary Ammonium). The upper limit concentration of acetone precipitation is preferably 80% by volume or less, more preferably 60% by volume or less, extremely preferably 40% by volume or less, and most preferably 30% by volume or less.

  The ice crystallization inhibitor according to the present invention may be extracted from a plant as it is, or may be extracted after inducing an ice crystallization inhibitor in a plant by a method such as low-temperature acclimation.

  The temperature for the low temperature acclimation is not particularly limited, but the lower limit temperature is preferably 0 ° C. or higher, and the upper limit temperature is preferably 20 ° C. or lower. The period of low temperature acclimation is not particularly limited, but it is preferably 3 days or more.

  The ice crystallization inhibitor according to the present invention can be easily extracted, purified and recovered.

  The extraction method is not particularly limited, and for example, it can be obtained by a known extraction method using water or an organic solvent.

  The form of the plant used for extraction is not particularly limited, and may be the whole seed and plant body, for example, a part of seed, bud, leaf, petiole, etc.

  Although the solvent for extracting the ice crystallization inhibitor is not particularly limited, it is preferably combined with at least one solvent selected from the group consisting of water, hydrophilic organic solvent, supercritical carbon dioxide, subcritical water and the like. Can be used.

  Examples of the hydrophilic organic solvent include methanol, ethanol, and the like, but those that can be used for food processing are preferable, and ethanol and the like are exemplified.

  Among these solvents, water and ethanol are preferable. It is also possible to use a mixture of water and an organic solvent.

  When using water, it is preferable to use heated water, particularly hot water, and when using an organic solvent, it is preferable to use a heated organic solvent.

  The temperature of the heated water or organic solvent is not particularly limited. The lower limit is preferably 0 ° C. or higher, and more preferably 20 ° C. or higher. The upper limit is preferably 160 ° C. or lower, and more preferably 120 ° C. or lower. In addition, examples of the aqueous solvent include various buffer solutions such as sodium acetate, and mixed solvents of alcohol and water, but are not limited thereto. The type and amount of the extraction solvent can be appropriately selected depending on the type and amount of the plant body to be extracted.

  Although it does not specifically limit as a purification method, For example, reverse osmosis, ultrafiltration, microfiltration etc. can be used combining suitably.

  The molecular weight cutoff for membrane separation is not particularly limited. The lower limit of the molecular weight cut off when the target product is recovered in the membrane-impermeable material is preferably 5,000 or more, more preferably 8,000 or more, extremely preferably 10,000 or more, and most preferably 15,000 or more. As long as the upper limit does not exceed 19,000, it can be suitably used.

  In the membrane separation method, a component having a low molecular weight selectively permeates through the membrane, and as a result, purification and concentration of a component having a high molecular weight in the solution is achieved. The permeation performance decreases with time due to accumulation (concentration polarization), adsorption onto the membrane surface and pores, and the like.

  When the ice crystallization inhibitor of the present invention is recovered on the polymer side, if the molecular weight cut off of the membrane used is less than 5,000, the removal of contaminating components in the solution is insufficient, and the membrane is clogged. Since it becomes easy, it is not preferable. Further, when the molecular weight cut off exceeds 19,000, purification and recovery of the ice crystallization inhibitor having a molecular weight of about 19 kDa become substantially difficult.

  The ice crystallization inhibitor of the present invention may be further purified as necessary. For example, contaminant components may be removed by suitably combining decantation, filtration, centrifugation, and the like. In addition, for example, salting out, precipitation with an organic solvent, affinity chromatography, ion exchange column chromatography, purification by gel filtration or the like, and concentration by dialysis or ultrafiltration may be suitably combined.

  Further, if necessary, it may be solidified into an arbitrary form such as powder or granule. The solidification method is not particularly limited. For example, the above-described extract is powdered according to a conventional method such as spray drying or freeze-drying, or the extract is adsorbed and supported on an excipient to form powder or granules. Examples of the method include solidification. These operations are known to those skilled in the art, and can be appropriately selected and used according to the application.

  The ice crystallization inhibitor according to the present invention can be used for the purpose of suppressing this failure in various fields in which failure occurs due to ice crystallization of water. For example, it can be used in the food field, machine field, civil engineering field, cosmetics field, medical field using biological samples, and the like.

  In the food field, it is possible to prevent deterioration of the taste of the food by suppressing the ice crystallization of water contained in the food. For example, it prevents starch aging, or suppresses deterioration of taste and quality caused by changing the structure of water in foods by crystallizing ice and physically pressing proteins and oil components. be able to.

  In the mechanical field and civil engineering field, it can be used as an antifreezing agent for moving parts, roads, ground, etc. of machines.

  In the cosmetics field, it can be used as an additive for preventing deterioration of cosmetic quality. For example, if a cosmetic containing an oil / fat component is frozen, water contained in the cosmetic may crystallize in ice, and the oil / fat component may be physically pressed to break the structure, thereby deteriorating the quality and feeling of use. If the ice crystallization inhibitor according to the present invention is used, the structure of the oil and fat component is maintained by preventing ice crystallization of water, so that deterioration of quality and the like can be suppressed.

  In the medical field, it can be used as a protective agent when cryopreserving a biological sample. For example, when a biological sample such as a tissue such as a cell, blood, or organ is frozen in a conventionally known preservation solution, the water in the preservation solution is frozen to produce ice crystals, which are damaged by the ice crystals. Sometimes. However, if the extract according to the present invention is added, the generation and growth of ice crystals can be suppressed, so that the biological sample can be protected from damage due to ice crystals.

  The form of the ice crystallization inhibitor of the present invention varies depending on its use, and may be a solution, a concentrated solution, a suspension, a lyophilized product, a powder, a granule, a tablet, or the like as it is.

  The antibody according to the present invention specifically reacts with the ice crystallization inhibitor and / or the polypeptide, and tests for the presence or absence of the ice crystallization inhibitor and the polypeptide in the plant. It can be used to identify the ice crystallization inhibitor and polypeptide from the mixture.

  The antibody according to the present invention may be prepared by a conventional method. For example, mice, rats, etc. are immunized using the above-mentioned ice crystallization inhibitor and polypeptide, and antibody-producing cells, spleen cells and myeloma cells are fused to obtain hybridomas. The hybridoma is cloned, and a clone producing an antibody that specifically reacts with the ice crystallization inhibitor or polypeptide is screened. This clone may be cultured and the secreted monoclonal antibody may be purified.

  Next, the method for measuring the activity of the ice crystallization inhibitor and the method for measuring the protein amount according to the present invention will be described.

  As the method for measuring the ice crystallization inhibitory activity of the ice crystallization inhibitor of the present invention, an appropriate method is used according to the type of plant. For example, it can be performed by a known method such as measurement of thermal hysteresis, observation of ice crystal structure, measurement of ice crystallization inhibitory activity, etc., when improvement of ice crystallization inhibitory activity is recognized by any method, It is intended to be included in the scope of the present invention. The ice crystallization inhibitory activity is measured, for example, by cooling the plant extract solution containing 30 w / v% sucrose to −40 ° C., then raising the temperature to −6 ° C., and measuring the average area of ice crystals observed with a microscope This can be done. The stronger the ice crystallization inhibitory activity is, the smaller the average area of the ice crystals is. Therefore, the ice crystallization inhibitory activity of the plant extract can be quantitatively evaluated using this value as an index. Compared to the control, if the formation of ice crystals is inhibited even when an ice crystallization inhibitor is added, it is judged to have ice crystallization inhibitory activity.

  The method for measuring the amount of protein in the ice crystallization inhibitor of the present invention is not particularly limited, and is performed by a known method such as the Lowry method, the bicinchoninic acid (BCA) method, the Bradford method (Coomassie method), or the like. be able to. Although it does not specifically limit as a standard protein, For example, bovine serum albumin (BSA) can be used suitably.

  Examples will be shown below, and the embodiments of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail. Further, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims, and the present invention is also applied to the embodiments obtained by appropriately combining the disclosed technical means. It is included in the scope of the invention. Moreover, all the patent documents and nonpatent literatures described in this specification are used as reference in this specification.

Production Example 1 Extraction 300 mg of dried radish extract was dissolved in 60 ml of Tris-HCl (5 mM, pH 8.0) to obtain a crude extract.

Production Example 2 Centrifugation 60 ml of the crude extract obtained in Production Example 1 was placed in a 300 ml beaker, heat-treated at 50 ° C. for 30 minutes using a water bath (manufactured by EYELA), and then centrifuged for 15 minutes ( 10,000 × g). The supernatant after centrifugation was collected.

Production Example 3 Acetone Fraction Acetone cooled to −30 ° C. was dropped little by little into 56 ml of the supernatant obtained after centrifugation obtained in Production Example 2, and the addition was continued until the final concentration reached 30% by volume. Centrifugation was performed for 15 minutes (10,000 × g), and the precipitate was collected. The resulting precipitate was lyophilized and dissolved in 6 ml of Tris-HCl (5 mM, pH 8.0).

Production Example 4 Ion Exchange Column Chromatography The concentrated solution obtained in Production Example 3 was diluted with 50 ml of Tris-HCl buffer (10 mM, pH 8.0). Charge 50 ml of this solution to a DEAE column (1.6 × 10 cm, manufactured by GE Healthcare) equilibrated with the same buffer, apply a 0-0.5 M NaCl gradient, and elute under conditions of a flow rate of 5 ml / min. The adsorbed fraction was collected.

Production Example 5 Gel Filtration Column Chromatography 52 ml of the DEAE non-adsorbing active fraction obtained in Production Example 4 was replaced with 2 ml of Tris-HCl (5 mM, pH 8.0). This solution was charged into a gel filtration column (Superdex200, manufactured by GE Healthcare) and eluted under conditions of a flow rate of 1.3 ml / min. As a result, a protein having a peak around 25 kDa was obtained.

Test Example 1 Measurement of Protein Concentration and Ice Crystallization Inhibitory Activity For each solution of Production Examples 1 to 4, the protein concentration and ice crystallization inhibitory activity were measured.

(1) Measurement of protein concentration The BCA method was used.

(2) Measurement of ice crystallization inhibitory activity Sucrose was added to each solution of Production Examples 1 to 4 at a rate of 30 w / v%. The average area of ice crystals observed with a microscope for 30 minutes while the temperature of the solution was raised to −6 ° C. and kept at −6 ° C. under a microscope having a stage with a cooling control function. Was measured. As a control, a 30 w / v% sucrose solution was measured in the same manner. The results are shown in Table 1. The values in the table indicate relative areas when the control ice crystal area is 1.0, and the smaller the average area of the ice crystals, the stronger the ice crystallization inhibitory activity.

  From Table 1, the activity per protein of Production Example 4 is higher than that of Production Example 1, and it is clear that the ice crystallization inhibitor was concentrated.

Test Example 2 Molecular Weight Measurement by SDS-PAGE The DEAE column chromatography active fraction of Production Example 4 was electrophoresed at 20 mA for 90 minutes using SDS-polyacrylamide gel (12.5% gel, manufactured by Ato). The gel after electrophoresis was stained with silver to visualize protein bands. From the result of gel staining, it was confirmed that proteins of about 19 kDa, about 22 kDa and about 59 kDa were contained.

Test Example 3 Determination of Amino Acid Sequence 20 μl of a solution containing 50 μg of the purified silkworm antifreeze protein obtained in Test Example 2 and 20 μl of sample buffer (EzApply, manufactured by ATTO) (solubility ratio 1: 1) are mixed, and 99 ° C. For 3 minutes. The sample was applied to 15% polyacrylamide gel (manufactured by ATTO, e-Pagel), and SDS-PAGE was performed at 20 mA for 90 minutes. The gel after SDS-PAGE was transferred to a PVDF membrane (Millipore, Immobilon PSQ) by a semi-dry method, and CBB staining was performed. The stained spot was cut out and the N-terminal amino acid sequence was determined by Edman method (manufactured by Shimadzu Corporation, protein sequencer PPSQ-33A). The resulting sequences were as follows:

SEQ ID NO: 1 (approximately 19 kDa): Gly-Phe-Glu-Ser-Thr-Lys-Cys-Met-Cys-Thr
SEQ ID NO: 2 (approximately 22 kDa): Met-Ala-Lys-Glu-Ala-Gln-Lys-Cys-Gln-Cys

  On the other hand, a sequence of about 59 kDa protein could not be obtained. This was thought to be because the N-terminus of the protein had undergone some modification.

Claims (12)

An ice crystallization inhibitor, which is derived from a plant and contains at least one protein of any one of (1) to (4) below.
(1) A protein having the amino acid sequence of SEQ ID NO: 1 and having a molecular weight of 19,000 ± 200 Da measured by SDS-PAGE (2) It has an amino acid sequence of SEQ ID NO: 2 and measured by SDS-PAGE Protein with a molecular weight of 22,000 ± 250 Da (3) Protein with a molecular weight of 59,000 ± 600 Da measured by SDS-PAGE and a modified N-terminus thereof (4) Above (1) to (3 A protein in which one or more amino acids of the amino acid sequence of any one of (2) is deleted, substituted or added, and has an activity to inhibit ice crystallization   2. The ice / ice crystallization inhibition according to claim 1, wherein the plant is one or more plants selected from the group consisting of Brassicaceae, Apiaceae, Lilyaceae, and Asteraceae, or a similar or improved variety thereof. material.   The plant belonging to the Brassicaceae family is one or more plants selected from the group consisting of Chinese cabbage, Japanese radish, broccoli, Chingensai, Komatsuna, turnip, Sirona, Nozawana, Hiroshima rape, Mizuna and mustard, or their related varieties or improved varieties The ice crystallization inhibitor according to claim 2, wherein   4. The ice crystallization inhibitor according to claim 3, wherein the plant belonging to the Brassicaceae family is radish (Raphanus sativus), or a similar or improved variety thereof.   5. The ice crystallization inhibitor according to claim 4, wherein the plant belonging to the Brassicaceae is silkworm radish.   The ice crystallization inhibitor according to any one of claims 1 to 5, which is contained in a fraction precipitated in an acetone fraction at an acetone concentration of 30% by volume.   A polypeptide obtained by dissociating the ice crystallization inhibitor according to any one of claims 1 to 6 and having an ice crystallization inhibitory activity.   An antibody characterized by specifically reacting with the ice crystallization inhibitor according to any one of claims 1 to 6 and / or the polypeptide according to claim 7.   A composition comprising the ice crystallization inhibitor according to any one of claims 1 to 6 and / or the polypeptide according to claim 7.   A food comprising the ice crystallization inhibitor according to any one of claims 1 to 6 and / or the polypeptide according to claim 7.   A biological sample protective agent comprising the ice crystallization inhibitor according to any one of claims 1 to 6 and / or the polypeptide according to claim 7.   A cosmetic comprising the ice crystallization inhibitor according to any one of claims 1 to 6 and / or the polypeptide according to claim 7.