JP2005239885A - Cooling liquid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cooling liquid not requiring a large-sized apparatus on use, having small influence on human bodies and environment when used and when discarded and causing little contamination to the apparatus. <P>SOLUTION: This cooling liquid comprises water and an antifreezing protein and has a freezing point lower than that of water. Since heat conductivity of the cooling liquid containing water and the antifreezing protein is higher than that of a cooling liquid containing glycols, or the like, a heat exchanger used when the cooling liquid containing water and the antifreezing protein is used as a cooling liquid for engines can be down-sized and reduction of size of whole apparatus and reduction of cost required for apparatus are made possible. Since COD (chemical oxygen demand) value of the cooling liquid containing water and the antifreezing protein is smaller than that of the cooling liquid containing glycol, or the like, influence on the environment when discarded is small. Further, the antifreezing protein has no toxicity to human bodies. Since freezing point of water is lowered not by salts, or the like, but by the antifreezing protein, contamination of the apparatus is reduced. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a coolant for cooling various devices such as an internal combustion engine.

  Conventionally, various coolants are known for cooling various devices such as an internal combustion engine. Among these, as a coolant for cooling an automobile engine, one containing water and alcohols or glycols as a freezing point depressant (melting point depressant) is generally used. Since the freezing point of the cooling liquid is lower than that of water due to alcohols and glycols contained in the cooling liquid, freezing of the cooling liquid in winter is prevented (for example, Patent Document 1).

  As alcohols and glycols as freezing point depressants, it is common to select ethylene glycol or propylene glycol, but ethylene glycol and propylene glycol have a lower thermal conductivity than water, so the engine can be cooled sufficiently. In order to do so, it is necessary to use a large amount of coolant. Furthermore, since the temperature of the coolant once raised by cooling the engine is difficult to decrease, it is necessary to provide a heat exchanger such as a large radiator in order to reduce the temperature of the coolant again. For these reasons, when ethylene glycol, propylene glycol, or the like is used as the freezing point depressant, there is a problem that the entire apparatus becomes large and the cost required for the apparatus increases.

  Moreover, since ethylene glycol and propylene glycol have a large COD (chemical oxygen demand), the environmental load at the time of disposal is large. Therefore, when the cooling liquid containing ethylene glycol or propylene glycol is discarded, it is generally recovered and incinerated, and there is a problem that the cost required for the disposal process becomes high and the disposal process is complicated. .

Furthermore, as a cooling liquid for a cooling device for cooling or freezing, an aqueous solution of a salt such as calcium chloride or salt commonly used as a brine is used, but the mass and viscosity increase because a large amount of the salt is added. As a result, there is a problem that the burden on the apparatus becomes large. In addition, when a cooling liquid composed of such an aqueous salt solution is used, the apparatus may be contaminated by precipitation of salts or oxidation of the apparatus. In this case, if the contamination of the apparatus progresses, the heat exchanger or the like is clogged, so it is necessary to add a large amount of anticorrosive additive, which increases the cost and increases the mass and viscosity, further increasing the burden on the apparatus. There was also a problem. For these reasons, there has been a demand for the development of a cooling liquid that is less contaminated and less burdened on the apparatus.
JP-A-7-70558

  It is an object of the present invention to provide a coolant that does not require a large-scale device during use, has a small influence on the human body and the environment during use and disposal, and is less contaminated and burdened on the device.

  The cooling liquid of the present invention that solves the above-mentioned problems includes water and antifreeze protein, and has a freezing point lower than that of water.

  The antifreeze protein preferably consists of the amino acid sequence represented by SEQ ID NO: 1.

  The antifreeze protein preferably consists of the amino acid sequence represented by SEQ ID NO: 2.

  The antifreeze protein is preferably contained in an amount of 0.01% by weight or more based on the total amount of the coolant.

  Antifreeze protein is a protein having the action of lowering the freezing point of water, and what is known from fish, insects, plants, bacteria, etc., so-called AFP (Antifreeze protein) or AFGP (Antifreeze glycoprotein) is well known. .

  The mechanism by which antifreeze protein lowers the freezing point of water is not yet clear, but it is thought that the freezing point of water drops because the growth of ice crystals is suppressed by the contact of antifreeze proteins with the surface of ice crystals. Yes.

  The cooling liquid of the present invention contains this antifreeze protein and water, and the freezing point of water is lowered by the antifreeze protein. Accordingly, even when the coolant of the present invention is used as a coolant for cooling an automobile engine, for example, the coolant is prevented from freezing in winter.

  Further, since the cooling liquid of the present invention does not contain ethylene glycol, propylene glycol, or the like, there is no increase in the size of the apparatus or increase in the cost required for the apparatus due to the low thermal conductivity of ethylene glycol or propylene glycol.

  And since this antifreeze protein reduces the freezing point of water sufficiently with a small amount, the COD value is very low and the influence on the environment is small, so there is no need to collect and incinerate at the time of disposal.

  Furthermore, according to the cooling liquid of the present invention, since the freezing point of water is lowered by antifreeze protein instead of salts, the contamination of the apparatus and the increase in mass and viscosity due to precipitation of rust and the occurrence of rust like conventional brine etc. There is no burden on the equipment.

  The coolant according to the present invention contains water and antifreeze protein. As the antifreeze protein, a protein that lowers the freezing point of water can be used.

  Antifreeze proteins are known to have a helix structure such as an α-helix structure or a β-helix structure, and antifreeze proteins are thought to exhibit excellent freezing point lowering effects by having these helical structures. . The α helix structure is a helical structure that rotates once for every 3.6 amino acid residues. Among antifreeze proteins having such an α helix structure, as shown in FIG. 1, threonine 1 is present every 10 amino acids. And the threonine 1 arranged in the same direction with respect to the helix axis (helical axis) 2 of the α helix structure is known to have a particularly large freezing point lowering effect.

  As the antifreeze protein, for example, a protein extracted or purified from fish or insect, which is generally known as the above-mentioned AFP or AFGP, can be used, or a synthesized antifreeze protein can also be used. Moreover, the antifreeze protein produced | generated by bacteria etc. can also be used by introduce | transducing the gene which codes these antifreeze proteins into bacteria etc. Furthermore, peptides extracted from active sites in these antifreeze proteins can be used as antifreeze proteins, and the precursors of these antifreeze proteins and these antifreeze proteins can be modified appropriately to improve heat resistance and structural stability. It is also possible to use derivatives having improved various properties such as properties.

  In addition, a buffer solution for stabilizing the three-dimensional structure of the antifreeze protein, an antibacterial agent for preventing the growth of microorganisms, and the like can be further added to the cooling liquid of the present invention. As the buffer solution, a general buffer solution having a pH of about 4.0 to 12.0 can be used. For example, Good's buffer solution, phosphate buffer solution, citrate buffer solution and the like are preferably used. Moreover, sodium azide etc. are preferably used as an antibacterial agent.

Hereinafter, the cooling liquid of the present invention will be described by way of examples.

(1. Selection of amino acid sequence of antifreeze protein)
Among the AFPs derived from the insect larvae of the yellow mealworm, the amino acid sequence represented by SEQ ID NOs: 3 to 12 is considered to be an active site that lowers the freezing point of water (Liou YC). , Et al (1999) Biochemistry (35): 11415-24. A complex family of high heterogeneous and antibacterial antifreeze. The common part of each amino acid sequence shown by sequence number 3-12 was extracted, and the amino acid sequence represented by sequence number 1 was obtained by arranging the extracted amino acid sequence part according to the order of the amino acid sequence. Specifically, in the amino acid sequences of SEQ ID NOs: 3 and 4, the 28th to 31st and 49th to 74th portions are represented; in the amino acid sequences of SEQ ID NOs: 5-8 and 10-11, the 28th to 62nd portions are represented; In the amino acid sequence of SEQ ID NO: 9, the 28th to 46th and 71st to 86th portions are compared, and in the amino acid sequence of SEQ ID NO: 12, the 21st to 55th portions are compared, and an amino acid having a high appearance frequency is selected and sequenced The amino acid sequence of number 1 was obtained. In addition, since it is known that a protein with an N-terminal amide having an α-helical structure, aspartic acid was introduced into the N-terminal of the amino acid sequence of SEQ ID NO: 1.

  Hereinafter, the antifreeze protein consisting of the amino acid sequence represented by SEQ ID NO: 1 is referred to as a first antifreeze protein.

  In addition, the amino acid sequence represented by SEQ ID NO: 2 is considered to be an active site that lowers the freezing point of water among the AFP derived from the fish flounder (Zang, W. et al ( 1998) J. Biol. Chem. 273, 34806-34812). Hereinafter, the antifreeze protein consisting of the amino acid sequence represented by SEQ ID NO: 2 is referred to as a second antifreeze protein.

(2. Synthesis of antifreeze protein)
Using a peptide synthesizer, the first antifreeze protein and the second antifreeze protein were synthesized according to the solid phase synthesis method by the Fmoc method described below. As a peptide synthesizer, an automated peptide synthesizer (Peptron III-R24, Peptron, Dajeon, Korea) was used. In addition, as an amino acid used at the start of the synthesis reaction, an amino group which is bonded to a resin via a carboxyl group and whose amino group is protected with a Fmoc (9-fluorenylcarbonyl) protecting group (Fmoc-amino acids and resins: Advanced) ChemTech, Louisville, USA). Unless otherwise specified, functional groups other than amino groups and carboxyl groups of amino acids are also protected with protecting groups.

  (1) The Fmoc protecting group of the amino acid which was bonded to the resin via the carboxyl group and the amino group was protected with the Fmoc protecting group was removed to liberate the amino group.

  (2) The amino acid was peptide-bonded to the free amino group by dehydration condensation of the carboxyl group of the amino acid whose amino group was protected with the Fmoc protecting group.

  (2) The Fmoc protecting group was removed from the resulting peptide.

  (3) The carboxyl group of another amino acid whose amino group was protected with an Fmoc protecting group was dehydrated and condensed with the free amino group.

  (4) The peptide chain was extended by repeating (2) to (3) by attaching amino acids one by one from the carboxyl terminal (C terminal) side to the amino terminal (N terminal) side.

  (5) All protecting groups were removed from the extended peptide chain. By the operations (1) to (5), a peptide mixture containing the target peptide and impurities was obtained.

  (6) The peptide mixture obtained in (5) was subjected to reverse phase chromatography (Waters 2690 Separations Module, Waters, Milford, USA, Waters C18RP column) and purified by elution with a solvent containing trifluoroacetic acid to remove impurities. It was. After that, it was dried by lyophilization (or reduced pressure drying) and crystallized.

  The target peptide was obtained by the operations (1) to (6), and the first antifreeze protein and the second antifreeze protein were synthesized. In addition, the kind of amino acid sequentially combined in the operations (1) to (4) is according to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

(3. Freezing point measurement test)
The first antifreeze protein synthesized in the above (2. Synthesis of antifreeze protein) is dissolved in water, and the coolant of Example 1 containing 0.01 wt% of the first antifreeze protein, the first A cooling liquid of Example 2 containing 0.05% by weight of the antifreeze protein and a cooling liquid of Example 3 containing 0.1% by weight of the first antifreeze protein were prepared.

  Similarly, the coolant of Example 4 containing 0.01% by weight of the second antifreeze protein and the coolant of Example 5 containing 0.05% by weight of the second antifreeze protein were prepared. 20 mg of each of the obtained cooling liquids of each example was taken, and the freezing point was measured by a differential scanning calorimetry method using DSC3100S (manufactured by Material Analysis and Charactorization).

  The graph showing the freezing point of the cooling fluid of Examples 1-5 measured by this freezing point measurement test is shown in FIG. In FIG. 2, the horizontal axis indicates the concentration of antifreeze protein (% by weight) contained in each cooling liquid, and the vertical axis indicates the value obtained by subtracting the freezing point of each cooling liquid from the freezing point of water (freezing point depression degree).

  As shown in FIG. 2, the cooling liquid of the present invention, which is an antifreeze protein aqueous solution, has a positive freezing point depression value, and the freezing point is lower than that of water. For this reason, it turns out that the cooling fluid of this invention is preferably used for the cooling fluid for motor vehicle engines, etc.

  For reference, FIG. 3 shows a graph showing the freezing point of an AFP aqueous solution derived from the larvae of the white rice beetle. This graph is shown in Timothy, S .; B. (Timothy, SB et al (1986) J. Biol. Chem. 261, 6890-6897).

  According to the graph shown in FIG. 3, the freezing point depression degree of the 0.01 wt% AFP aqueous solution is 1.2 ° C. On the other hand, according to the graph shown in FIG. 2, the freezing point depression degree of the antifreeze solution of Example 1 containing 0.01% by weight of the first antifreeze protein is 4.5 ° C., which is shown in FIG. It can be seen that the freezing point is significantly lower than that of the AFP aqueous solution. This is due to the fact that the first antifreeze protein is obtained by extracting only the active site from AFP. That is, in the case of the same weight, the first antifreeze protein consisting only of the active site has more active sites than AFP including a portion other than the active site. For this reason, it is considered that the first antifreeze protein aqueous solution has a higher freezing point depression degree than the normal AFP aqueous solution.

  In the cooling liquid of the present invention, for example, not only the antifreeze protein consisting only of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, but also one or several amino acids are included in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. Those consisting of amino acid sequences deleted, substituted and / or added can also be used. In this case as well, the more the active site, that is, the portion of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is contained relative to the other portion, the greater the degree of freezing point depression.

(4. Thermal conductivity measurement test)
A cooling liquid of Comparative Example 1 containing 50% by weight of ethylene glycol was prepared. The thermal conductivity of the coolant of Comparative Example 1 and the coolant of water was measured by the unsteady hot wire method.

  The graph showing the heat conductivity in 20 to 80 degreeC of the water measured by this heat conductivity measurement test and the cooling fluid of the comparative example 1 is shown in FIG. In FIG. 4, the horizontal axis indicates temperature, and the vertical axis indicates thermal conductivity.

  As shown in FIG. 4, water showed a larger thermal conductivity than the cooling liquid of Comparative Example 1 which is a conventional cooling liquid. In addition, since the cooling liquid of each Example which is a cooling liquid of this invention melt | dissolves or disperses a trace amount protein in water, thermal conductivity is substantially the same as water.

  Therefore, when the coolant of the invention is used as a coolant for an engine of an automobile, the engine can be sufficiently cooled with a small flow rate, and the load applied to the pump for circulating the coolant is reduced. Further, since the coolant of the present invention has a large thermal conductivity, the temperature of the coolant once raised by cooling the engine is easily lowered. Therefore, a small heat exchanger for cooling the coolant is sufficient, and the entire apparatus can be downsized and the cost required for the apparatus can be reduced.

(5. COD test)
Comparative Example 2 containing 10% by weight of ethylene glycol, Cooling liquid of Comparative Example 3 containing 12% by weight of ethylene glycol, Cooling liquid of Comparative Example 4 containing 16% by weight of ethylene glycol, Comparison containing 20% by weight of ethylene glycol The theoretical COD was calculated for the coolant of Example 5, the coolant of Comparative Example 6 containing 22% by weight of ethylene glycol, and the coolants of Examples 1-5.

The calculation method was based on the method described in the New Revised Environmental Analysis Technology Method (Edited by Japan Environmental Measurement Analysis Association, Shirakaba Publishing). The first protein is oxidized according to the reaction shown in Chemical Formula 1, and the second protein is oxidized according to the reaction shown in Chemical Formula 2. Therefore, to fully oxidize 10 ⁷ ppm of the first protein (molecular weight 3024), 19.7 × 10 ⁵ ppm of oxygen completely oxidizes the 10 ⁷ ppm of the second protein (molecular weight 3813). Is considered to require 17 × 10 ⁵ ppm of oxygen. In order to completely oxidize the coolant of Comparative Example 2 at 10 ⁷ ppm, 12.5 × 10 ⁴ ppm of oxygen is required to completely oxidize the coolant of Comparative Example 3 at 10 ⁷ ppm. × 10 ⁴ ppm of oxygen completely oxidizes the coolant of Comparative Example 4 with 10 ⁷ ppm 20.0 × 10 ⁴ ppm of oxygen completely oxidizes the coolant of Comparative Example 5 with 10 ⁷ ppm oxygen 25.6 × 10 ⁴ ppm in to is considered to be a necessary oxygen 28.16 × 10 ⁴ ppm is to completely oxidize the coolant of Comparative example 6 10 ⁷ ppm.

  FIG. 5 shows a graph representing the calculated theoretical COD values of the coolants of the examples and comparative examples. In FIG. 5, the vertical axis represents the theoretical COD value of each coolant, and the horizontal axis represents the freezing point depression degree of the coolants of the examples and comparative examples.

  As shown in FIG. 5, the theoretical COD value of the cooling liquid of the example is significantly smaller than that of the cooling liquid of the comparative example having the same freezing point depression degree. This is presumably because the amount of antifreeze protein added to the cooling liquid is small because the antifreeze protein exhibits an excellent freezing point lowering effect even in a small amount.

  Table 1 shows the freezing point depression degree, theoretical COD value, and thermal conductivity of the coolants of Example 3, Example 5, and Comparative Examples 2-6.

  As shown in Table 1, the coolants of Example 3 and Example 5 exhibit the same degree of freezing point depression as the coolants of Comparative Example 5 and Comparative Example 6. It shows a higher thermal conductivity than the coolant and a COD that is significantly smaller than the coolants of Comparative Examples 5 and 6. Also from this result, it can be seen that the coolant of the present invention is superior to conventional coolants in which glycols and the like are blended.

(6. Thermal stability test)
The second antifreeze protein synthesized in (2. Synthesis of antifreeze protein) was dissolved in water to prepare a cooling liquid of Example 6 containing 0.35% by weight of the second antifreeze protein. The coolant of Example 6 was placed in a thermostatic bath and heat-treated at 90 ° C. for 1 hour, 3 hours, and 6 hours.

  The freezing point of the coolant of Example 6 heat-treated for each time was measured by the same method as (3. Freezing point measurement test). Moreover, the freezing point of the cooling liquid of Example 6 which was not heat-processed was measured similarly. The residual activity of the cooling liquid after the heat treatment was calculated from the freezing point of the cooling liquid heat-treated for each time and the freezing point of the non-heat-treated cooling liquid. The residual activity was calculated by the following formula.

Residual activity (%) = freezing point of heat-treated cooling liquid / freezing point of non-heat-treated cooling liquid x 100
A graph showing the remaining activity of the coolant of Example 6 measured in the freezing point measurement test is shown in FIG. In FIG. 6, the horizontal axis indicates the time during which each coolant is subjected to heat treatment, and the vertical axis indicates the remaining activity (%) of the coolant.

  As shown in FIG. 6, the coolant of Example 6 containing the second antifreeze protein maintained 80% freezing point depressing activity before the heat treatment despite being heat treated at 90 ° C. for 6 hours. In general, proteins have a three-dimensional structure that changes at about 60 ° C, so that they do not function when exposed to high temperatures. However, the antifreeze protein of the present invention has excellent thermal stability and is exposed to constant temperatures such as automotive engine coolants. It can be seen that it is preferably used as a cooling liquid.

(7. pH stability test by simulation)
As described above, since it is considered that the helical structure of the antifreeze protein is a part that exhibits the freezing point depressing action, it is considered that the antifreeze protein having a lot of helical structure exhibits a more excellent freezing point lowering action.

  On the other hand, the amount of helical structure contained in a protein depends on pH. That is, since the tertiary structure of the protein changes depending on the pH to which the protein is exposed, the amount of the helical structure contained in the protein changes depending on the pH. Therefore, the amount of α-helix when the antifreeze protein of SEQ ID NO: 1 and the antifreeze protein of SEQ ID NO: 2 were exposed to various pHs was simulated. The simulation was performed using Agadir (http://www.embl-heidelberg.de/cgi/agadir-wrapper.pl), which is a peptide α-helix structure prediction software. Using this software, the amino acid sequences of the antifreeze proteins of SEQ ID NO: 1 and SEQ ID NO: 2 were input, and the α helix content (%) in the case of pH 1-14 under conditions of 278 K (4.85 ° C.) and ionic strength 0 ) The α-helix content was defined as the proportion of amino acids having a helix structure among all amino acids in the peptide.

  FIG. 7 shows the relationship between pH obtained by simulation and α-helix content. In addition, the vertical axis | shaft in FIG. 7 shows alpha helix content rate (%), and a horizontal axis shows pH.

  As shown in FIG. 7, it is estimated that the antifreeze protein of SEQ ID NO: 1 has a high α helix content in the range of pH 8 to 11, and the highest α helix content in the vicinity of pH 9.8. In addition, it is estimated that the antifreeze protein of SEQ ID NO: 2 has a high α helix content in the range of pH 7 to 14, and the highest α helix content in the vicinity of pH 8.2. Therefore, it is considered that the antifreeze protein of SEQ ID NO: 1 shows a better freezing point depressing action near pH 8-11, and the antifreeze protein of SEQ ID NO: 2 shows a better freezing point lowering action near pH 7-14.

  Here, the antifreeze liquid of each Example which measured the freezing point by the above-mentioned (3. Freezing point measurement test) was acidic at about pH 3.1-3.2. This is probably because trifluoroacetic acid was used as a solvent when the antifreeze protein was purified by reverse phase chromatography after the synthesis of the antifreeze protein. Most of the trifluoroacetic acid is volatilized when the antifreeze protein is freeze-dried or dried under reduced pressure after purification, but a part of it remains, so the pH of the antifreeze solution is considered to be low. Therefore, if the pH of the antifreeze solution of each Example is adjusted to about 8 to 11 for the antifreeze protein of SEQ ID NO: 1 and about 7 to 14 for the antifreeze protein of SEQ ID NO: 2, (3. Freezing point measurement test) It is considered that the freezing point is lowered more than the result obtained in the above.

It is a figure which represents typically the alpha helix structure of general antifreeze protein. It is a graph showing the freezing point of the cooling fluid of Examples 1-5 measured by the freezing point measurement test. It is a graph showing the freezing point of the AFP aqueous solution derived from the larvae of the white rice leafworm. It is a graph showing the heat conductivity in 20 to 80 degreeC of the water measured by the heat conductivity measurement test, and the cooling fluid of the comparative example 1. FIG. It is a graph showing the theoretical COD value of the cooling fluid of each Example and comparative example calculated by the COD test. It is a graph showing the residual activity of the cooling fluid of Example 6 measured by the freezing point measurement test. It is a graph showing the relationship between pH obtained by simulation, and alpha helix content rate.

Claims (4)

  A cooling liquid comprising water and antifreeze protein and having a freezing point lower than that of water.   The coolant according to claim 1, wherein the antifreeze protein has an amino acid sequence represented by SEQ ID NO: 1.   The coolant according to claim 1, wherein the antifreeze protein has an amino acid sequence represented by SEQ ID NO: 2.   The coolant according to claim 2 or 3, wherein the antifreeze protein is contained in an amount of 0.01 wt% or more based on the total amount of the coolant.

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