KR100532860B1 - Protein preserving medium comprising cathodic electrolyzed water and method of preserving protein using the same - Google Patents

Abstract

The present invention relates to a protein preservation solution containing cathodic electrolyzed water and a protein preservation method using the same. The protein preservation solution including the reduced ionization water according to the present invention can be applied as a reaction cleaning solution in the field of biotechnology as a simple, economical and environmentally friendly preservation solution that prevents degradation of the protein and contributes to the structural stabilization of the protein.

Description

Protein preserving medium comprising cathodic electrolyzed water and method of preserving protein using the same

The present invention relates to a protein preservation solution containing a reduced ionized water and a protein preservation method using the same.

Applying a direct voltage to the water creates electrolyzed water that can change pH by the movement of ions. The water produced at the anode decreases in pH due to the increase of H ⁺ ions, and the oxidation-reduction potential increases, resulting in a strong oxidative state. ^­ The pH rises due to the increase of ions and becomes a reducing state (Ryoo, K., Kang, B. and Sumida, S., Electrolyzed water as an alternative for environmentally-benign semiconductor cleaning. Material Research Society, 17 (6), pp. 1298-1304, 2002). The redox potential of ionized water shows a much stronger pH dependence than other aqueous solutions. In general, the redox potential of the ionized water at pH 2 shows a high oxidation potential of +1200 mV or more, while the acidic aqueous solution shows a redox potential of about +600 mV. The redox potential of the ionized water at pH 11 has a reduction potential of -850 mV or less, but +20 mV for alkaline aqueous solutions.

The characteristics of the ionizing water include bactericidal activity against microorganisms (Fabrizio, KA and Cutter, CN, Stability of electrolyzed oxidizing water and its efficacy against cell suspensions of Salmonella typhimurium and Listeria monocytogenes. J. Food Prot., 66 ( 8), pp. 1384-1397, 2003; Sharma, RR and Demirci, A., Treatment of Escherichia coli O157: H7 inoculated alfalfa seeds and sprouts with electrolyzed oxidizing water.Int. J. Food Microbiol., 86 (3), pp.231-237, 2003), and the treatment of wounds and burns in mice (Xin, H., Zheng, YJ, Hajime, N. and Han, ZG, Effect of electrolyzed oxidizing water and hydrocolloid occlusive dressings) on excised burn-wounds in rats.Chin.J. Traumatol., 6 (4), pp.234-237, 2003) It has been reported to have a positive effect on the treatment of diseases such as diabetes (Huang, KC, Yang, CC, Lee, KT and Chien, CT, Reduced hemodialysis-induced oxidative stress in end-stage renal disease patients by electrolyzed reduced water Kidney Int., 64 (2), pp.704-714, 2003). It has also been reported that cathodic electrolyzed water can inhibit oxidative damage of DNA because it has antioxidant activity (Sanetaka, S., Shigeru K., Mariko). , N., Takumi, M., Kenichi, K., Miho, G., Hidemitsu, H., Kazumichi, O., Shinkatsu, M. and Yoshinori, K., Electrolyzed-reduced water scavenges active oxygen species and protects DNA from oxidative damage.Biochemical and Biophysical Research Communications, 234, pp. 269-274, 1997). In addition, the ionized water by adjusting pH or redox potential can effectively remove impurities attached to the solid surface. By varying the electrolysis conditions and controlling the surface conditions such as hydrophilicity or lipophilic, it is possible to facilitate the treatment of the fine structure and the reactants, thereby creating an effective cleaning medium for various purposes (Kang, Byung-Doo. Semiconductor Cleaning Using Soonchunhyang University, pp.21-23, 2001).

On the other hand, the three-dimensional structure of a protein is maintained by the structure holding | maintenance force, such as the peptide bond between amino acid residues with strong binding force, and the ionic bond, hydrogen bond, and minor bond which are weak in binding force. For this reason, the higher-order structure of secondary or higher structure loses its structure holding power relatively easily by heat, acid, alkali, organic solvent, free radicals, etc. and cannot maintain its three-dimensional structure, and cannot decompose its original function.

Various studies have been conducted to improve the stability of proteins that are unstable to the external environment.

The protein stabilization method by the freeze-drying method is a process of subliming ice by applying vacuum in a frozen state of a protein solution and drying the protein without damaging the protein. Once successfully dried, most protein denaturation does not occur, so it is used as a method of preserving weak protein products for a long time. Specifically, a method using a solution in which 20 to 50% of glycerol or ethylene glycol is added at -20 ° C, a method of lyophilizing in liquid nitrogen at -20 ° C to -80 ° C, and the like are known. There is a problem in that a sterilization process or an antimicrobial agent is added or a freezing process is required, and when a protein is denatured during drying, it is difficult to reduce the titer when the solution is made again.

Suspension stabilization is not a commonly used method of protein stabilization, but it is a method that contributed to stabilizing insulin and completing a liquid product. Insulin is also known to be a protein that is difficult to store as it is classified as a very unstable protein, but metals such as zinc can be added to create a colloidal precipitate that can dramatically increase its stability. However, this method also requires that the precipitated protein particles dissolve into proteins that maintain their titer again after injection, but a significant number of proteins are inactivated and cannot be returned from the precipitation state, and the precipitated proteins cause side effects at the injection site or It has the disadvantage of being able to cause production and of being applied only to products of low protein concentration.

Generally, proteins that are easily degraded by oxidative modification are known. In the case of hemoproteins with heme in the structure, the degradation of proteins is accelerated. This is because active oxygen species are actively produced near Fe in the heme. The degradation of proteins by such oxidative degeneration is known to be inhibited by reactive oxygen species scavenger (Lee Mi Young, Kim Soung Soo, Inactivation and cleavage of radish peroxidase by various reducing agents.Phytochemistry, Vol. 49, No. 1 , pp. 23-27, 1998; Kim, KH, Rhee, SG and Stadtman, ER, Journal of Biological Chemistry, 1985, 260, 15394).

Generally, there is a method of using tertiary distilled water of 4 ° C. or lower as a medium for preserving protein, but in this case, the titer of the protein has a disadvantage in that it is easily denatured even under minute conditions as it depends on the flowable tertiary structure. In particular, protein aggregation due to oxidative degeneration has been pointed out as a problem. In order to solve the above problems, an additive such as sugars, amino acids, or salts may be used, but the concentration of the additive is often so high that it is unsuitable for use in the intended use, and its application is limited.

On the other hand, since the ionized water exhibits acidic (acidic ionized water) or alkaline (reduced ionized water), it is expected to decompose the protein by giving structural instability to proteins sensitive to acid and alkali. Contrary to these expectations, however, it has been found that the reduced ionizing water does not affect the structural stability of the protein and thus exhibits an excellent protection against protein degradation and thus can be used as an economical and environmentally friendly protein preservative. Came to complete.

It is an object of the present invention to provide a protein preservation solution having excellent protein preservation ability and being environmentally friendly and economical and a protein preservation method using the same.

The present invention provides a protein preservation solution including reduced ionized water.

As used herein, the term "electrolyzed water" is an aqueous solution in which pH or oxidation-reduction potential is adjusted by electrolysis. Electrolysis of water is one of the electrochemical phenomena, which changes the properties of water to provide new chemicals, and ionizing alkaline water (cathode ionizing water) and ionizing water, which are different ionizing products at the anode and the cathode. Create an anode ionizer. Electrolysis phenomena can be handled quantitatively and are used in a variety of applications in the industry. As used herein, "cathodic electrolyzed water" means reduced water collected from a cathode when electrolyzing water.

The reduced ionized water contained in the protein preservation solution of the present invention can be prepared using various apparatuses and known methods for electrolyzing water. The ionizer generator for generating ionized water has an electrolytic cell divided into two areas in which an anode and a cathode are disposed by a separator. When an electric current flows through the anode, ionized alkaline water is generated at the cathode and ionized at the anode. Acidic water is produced. Typically, a voltage of at least 2 to 3 volts or higher is supplied, a diaphragm or ion exchange membrane is installed to separate the anode and the cathode, and an electrolytic material (eg, NaCl, KCl, etc.) is added to promote electrolysis. . Many devices are commercially available for producing electrolyzed tap water by directly electrolyzing tap water. The continuous generation of ionizing water is filtered by tap water using an adsorption medium such as activated charcoal, and then purified water is continuously supplied to the electrolytic cell so that the ionizing water is continuously generated. In addition, batch devices for producing ionized water comprising an anode and a cathode chamber are also well known.

Apparatuses for producing reduced water for various applications have been researched and developed. As a specific example, after supplying pure water to an electrolyte preparation tank, an electrolyte having a predetermined molar concentration of NH ₄ OH or HCl is prepared, and then an anode electrode. By applying a voltage having a predetermined magnitude between the cathode and the cathode, electrolytic solution of the electrolytic cell may be electrolyzed to produce reduced water having a desired pH from the cathode. In addition to the above-described method, a device capable of continuously generating a large amount of ionized water with high efficiency, comprising a flow restricting means in an electrolytic cell so that the raw water can maintain a long contact between the negative electrode plate and the positive electrode plate. BACKGROUND OF THE INVENTION There are known ionizing water generating apparatuses which have a very narrow interval.

In the present invention, the term "protein" refers to an organic polymer produced by dehydration condensation of a carboxyl group of an amino acid and an amino group of another amino acid, and refers to a molecule including an oligopeptide and a polypeptide. The protein can be classified into simple protein, complex protein and derived protein according to chemical composition or solubility. This includes, but is not limited to:

(a) simple protein

Refers to a protein that generates only amino acids when hydrolyzed, and is an albumin (eg, serum albumin, lactalbumin, egg white albumin, etc.) present in animal and plant cells and body fluid as a protein that is dissolved in water and coagulated by heat; Globulins (serum globulin, β-lactoglobulin, etc.), most of which are proteins that are soluble in dilute salt solution without being dissolved in water; Glutenin and oryzenin, which are high in glutamic acid and are contained in grain seeds; Prolamine (gliadine, zein, etc.); Albuminoids (collagen, keratin, elastin and the like); Histones; Protamine.

(b) complex proteins

As a protein to which a simple protein and a cofactor bind, Nucleoproteins such as deoxyribonucleoprotein and ribonucleoprotein, which are complexes of DNA and protein; Phosphoproteins such as casein, vitellin, and phosvitin, in which the phosphoric acid is contained in ester form in the protein; Lipoproteins such as thromboplastin and lipoitellin, complexes of lipids and proteins; Metals directly bind to proteins, iron proteins containing heme, such as ferredoxin, transferrin, ferritin, platocyanin, copper proteins such as ascorbate oxidase, pyruvate carboxylase Metalloproteins such as manganese proteins such as; Glycoproteins such as avidin, mucin and thyroglobulin.

(c) inducing proteins

Intermediates during protein hydrolysis include proteases, peptones, dipeptides, tripeptides and the like.

In addition, the protein preservation solution according to the present invention has a primary structure in which amino acids form a long chain structure with peptide bonds, and peptide chains appear in a repetitive manner in which alpha-helix (helix) and beta-sheet (screen structure) are uniformly three-dimensionally. Tertiary structure consisting of hydrogen, hydrophobic, ionic and disulfide bonds between amino acids with secondary structures separated from one another, and proteins with quaternary structures in which these bonds are more intricately intertwined Can be used for the preservation of

In addition, the protein preservation solution according to the present invention is a therapeutically active protein, such as stabilizers, muscle relaxants, antidepressants, anti-inflammatory agents; It can be used for the preservation of proteinaceous hormones such as thyroid stimulating hormone, follicle stimulating hormone, growth hormone and the like and various other enzymes.

Especially preferred proteins for use of the protein preservative of the present invention include insulin, superoxide dismutase (SOD), interferon, growth hormone, peroxidase, asparaginase, glutamase, aginanase, somatomethine, somatostatin, vasopressin, RNA It is an unstable protein such as an enzyme, more preferably a protein that is easily degraded by oxidative denaturation, and most preferably a hemoprotein containing heme.

The redox potential and pH of the reduced ionization water are correlated and the reducing power is increased as the redox potential becomes negative. The pH is 6-7 when the redox potential is about -200 mV, and the pH is 8.5-10 when the redox potential is -800 mV. In the present invention, the redox potential of the reduced ionized water used for protein preservation is -200 mV or less, preferably -1000 mV to -200 mV, more preferably -900 mV to -800 mV, and most preferably -840 mV to -860 mV. , pH is 6 or more, preferably 6 to 11, more preferably 8.5 to 11, most preferably 10 to 11. When the redox potential of the reduced ionized water is -200 mV or more, the protein is decomposed to reduce the protein retention.

In order to investigate the effect of reducing ionization on the preservation of the protein, the inventors observed the degradation of the protein by varying the retention period and temperature.

Figure 1 shows the result of the protein decomposition after the reaction of the protein preservation effect of the acidic ionized water and reduced ionized water for 10 minutes. The protein used was horseradish peroxidase, which was dissolved in tertiary distilled water, acidic ionized water and reduced ionized water as a control, and reacted for 10 minutes, followed by electrophoresis. As a result, the protein reacted in the reduced ionized water did not decompose, whereas the protein dissolved in the tertiary distilled water formed about 26 kDa band, and the protein dissolved in the acidic ionized water also formed bands at about 26 kDa and 35 kDa.

FIG. 2 shows the results of observing the degree of degradation of the protein at 4 ° C. and 25 ° C., respectively, in order to examine the effect of temperature while maintaining the retention time as 7 days. After 7 days, the protein reacted with the reduced ionized water did not decompose, whereas the protein dissolved in the tertiary distilled water and the acidic ionized water reacted for 10 minutes. It was confirmed that the degradation of the protein proceeded further.

3 shows the results of observing the degree of degradation of proteins at 4 ° C. and 25 ° C., respectively, with a retention time of 10 days. Even after 10 days, protein degradation was not observed in the reduced ionized water at 4 ° C and 25 ° C. However, in the case of the tertiary distilled water at 4 ° C and 25 ° C, the protein band of about 26 kDa, which was initially decomposed, was maintained, and about 17 kDa including protein band of 26 kDa and 35 kDa in acidic ionized water. Protein bands of 22 kDa and 38 kDa were formed, and the protein bands reacted at 25 ° C were more degradable than those reacted at 4 ° C.

Through these results, it was confirmed that the protein reacted in the reduced ionized water was hardly decomposed without being affected by the temperature change during the entire reaction time. Therefore, the reduced ionized water has a strong protection against protein degradation and contributes to the structural stabilization of the protein, and can be used for long term, preferably at least 4 weeks, more preferably at least 10 days of protein preservation. Preferably it is 30 degrees C or less, More preferably, it is 25 degrees C or less, More preferably, it can be used for protein storage of 4 degreeC-25 degreeC.

As a further aspect of the present invention, the protein preservative of the present invention may further add protein inhibitors, metal chelates, dithiothreitol (DTT), reducing agents such as 2-mercaptoethanol, etc. to the reduced ionized water to increase the shelf life. .

Since protein preservatives of the present invention are excellent in protein preservation, biotechnology, such as ELISA (enzyme linked immunosorbent assay), Western blotting (western blotting), separation and purification of proteins, mass production of enzymes, etc. Can be used.

If the protein preserved in the reduced ionized water of the present invention is an enzyme, not only the protein should be degraded, but also it should not adversely affect subsequent enzymatic reactions. Table 1 and Table 2 of Example 2 below show the results of measuring the enzyme activity of horseradish peroxidase in reduced ionized water. The experimental results show that, unlike the acidic ionized water in which the activity of the enzyme is sharply reduced, the reduced ionized water of the present invention not only prevents the degradation of the enzyme protein but also shows an excellent effect of protecting the activity of the enzyme.

In still another aspect, the present invention provides a protein preservation method using reduced ionized water. As demonstrated from FIG. 1 to FIG. 3, the reduced ionized water prevented protein degradation and contributed to structural stabilization without affecting the storage period and storage temperature, and thus the protein preservation effect was excellent.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the examples.

Example 1 Protein Preservation of Reduction Ionized Water

To determine the effect on proteolysis, 1 mg of purified horseradish peroxidase (Sigma-Aldrich Co.) (40 kDa) was added to control distilled water, control acidic ionized water and reduced ionized water (ORP -850). mV, pH 11) completely dissolved in 1 ml and left for 10 days at 4 ℃ and 25 ℃. 25 μg of horseradish peroxidase at 1, 7, and 10 days intervals were collected from the control and control groups, respectively, to determine the degree of protein degradation. Stacking gel by SDS-polyacrylamide (Laemmli 1970) electrophoresis ) 5% and 12.5% separation gel (separating gel) was developed and compared, compared with the standard protein myosin (205 kDa), β-galactosidase (116 kDa), phosphorylase b (97 kDa), Fructose-6-phosphate kinase (84 kDa), albumin (66 kDa), glutamic dehydrogenase (55 kDa), egg white albumin (ovalbumin) (45 kDa), glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde) -3-phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), trypsin inhibitor (20 kDa), α-lactalbumin (14.2 kDa), apro A sigma marker mixed with aprotinin (6.5 kDa) was used.

When reacted for a short time of 10 minutes and electrophoresis, horseradish peroxidase dissolved in tertiary distilled water and acidic ionized water as a control, about 26 kDa band was formed as the protein began to decompose, The dissolved protein also began to degrade and a band was formed at about 26 kDa and 35 kDa, but the protein reacted in the reduced ionized water did not degrade (FIG. 1).

Proteins dissolved in the tertiary distilled water and the acidic ionized water for 7 days at 4 ° C. and 25 ° C., respectively, formed the same protein bands as the first ones, and the degree of degradation was remarkably increased (FIG. 2).

Proteins reacted at 10 days in tertiary distilled water at 4 ° C. and 25 ° C. conditions, respectively, retained a protein band of about 26 kDa that was initially degraded, and no further degradation occurred. In acidic ionized water, about 17 kDa, 22 kDa, 38 kDa protein bands were formed, including about 26 kDa and 35 kDa protein bands. It can be inferred that the higher the temperature, the proteolytic effect of the acidic ionized water may be affected by the temperature, and the protein reacted in the reduced ionized water was hardly decomposed without affecting the temperature change during the entire reaction time (Fig. 3). ).

These results suggest that acidic ionized water may induce degeneration or degradation of proteins in vitro , suggesting that prolonged exposure to acidic ionized water may denature animal tissue cells or proteins. On the other hand, the reduced ionized water was able to confirm the protective action against protein degradation unlike the distilled water and the acidic ionized water.

Example 2 Effect of Reduction Ionize on Enzyme Activity

In order to investigate the effect of the reduced ionization number on the enzyme activity, the enzyme activity of horseradish peroxidase was measured. In the 150 mM sodium phosphate buffer (pH 6.0), the final concentration of the first substrate, guaiacol, was 15 mM, and the final concentration of H ₂ O ₂ , the second substrate, was 5 mM. The amount of change in absorbance changed at 470 nm for 1 minute by enzymatic reaction by multiple agents was measured at 4 ° C. and 25 ° C., respectively (Table 1 and Table 2). The activity of an enzyme refers to the activity of a certain amount of enzyme protein.

Relative enzyme activity according to reaction time at 4 ℃ 4 ℃          Relative enzyme inactivity (%) 10 minutes 7 days 10 days 3rd distilled water 100 100 100 Acid ionized water 61.3 13.53 9.3 Reduction ionization 101.8 102.7 103.2

Relative enzyme activity with reaction time at 25 ℃ 25 ℃          Relative enzyme inactivity (%) 10 minutes 7 days 10 days 3rd distilled water 100 100 100 Acid ionized water 61.3 0 0 Reduction ionization 101.8 102.0 102.4

As can be seen from the above, the reduced ionized water showed an excellent protective effect similar to tertiary distilled water as well as protecting against enzyme protein degradation.

As described and demonstrated in detail above, the protein preservation solution using the reduced ionization water of the present invention prevents degradation of the protein and contributes to the structural stabilization of the protein, so that it is a simple, economical and environmentally friendly preservative solution, and can be applied as a reaction washing solution in the biotechnology field. Can be.

Figure 1 shows the results of electrophoresis on the degradation of protein after 10 minutes in order to analyze the protein preservation effect of acidic and reduced ionized water.

Figure 2 is the result of electrophoresis for the degradation of protein after 7 days in order to analyze the protein preservation effect of acidic and reduced ionized water.

Figure 3 is the result of electrophoresis of the degradation of the protein after 10 days in order to analyze the protein preservation effect of the acidic and reduced ionized water.

* Description of reference numerals of FIGS. 1 to 3

Lane 1: marker

Lane 2: 4 ° C tertiary distilled water

 Lane 3: acidic ionized water at 4 ℃

Lane 4: 4 ℃ reduced ionized water

Lane 5: 25 ° C tertiary distilled water

Lane 6: acidic ionized water at 25 ℃

Lane 7: 25 ℃ reduced ionized water

Claims (7)

Protein preservation solution containing reduced ionized water. The protein preservation liquid of Claim 1 whose redox potential of a reduced ionization water is -1000mV--200mV. The protein preservative of claim 1, wherein the pH of the reduced ionized water is 6-11. The protein preservative of claim 1, further comprising at least one selected from the group consisting of a protein inhibitor, a metal chelate, dithiothreitol (DTT), and 2-mercaptoethanol. Preservation of protein using reduced ionization water. The method according to claim 5, wherein the redox potential of the reduced ionization water is -1000 mV to -200 mV. The method of claim 5 wherein the pH of the reduced ionized water is 6-11.