JP5245053B2 - Tripeptide synthesis method, tripeptide synthase solution, and production method thereof - Google Patents

Description

The present invention relates to a tripeptide synthesis method, a tripeptide synthase solution, and a production method thereof, and more particularly, a useful tripeptide having a specific amino acid sequence in which β-alanine, ornithine and ornithine are peptide-bound in this order from the N-terminus. The present invention relates to a synthesis method, a tripeptide synthase solution used therefor , and a production method thereof .

  Proteins are substances that directly take charge of the structure and function of cells, but there are many types, and they exist everywhere in the cell nucleus and cytoplasm and perform their functions. Various enzymes, peptide hormones, antibodies, etc. are proteins and are important players in life phenomena. In addition, by using a specific reaction between a protein and a specific substance, it is possible to measure the target substance with high accuracy. Therefore, clinical diagnosis using food functions, food analysis, environment Analysis etc. are being conducted. At present, proteins are widely used. On the other hand, proteins are generally weak against heat, and when stored at room temperature, they often aggregate and a decrease in activity is observed. Therefore, there is a need for an effective protein stabilizer in order to use the protein in industry.

The present inventors have found that a tripeptide (hereinafter also referred to as “β-Ala-Orn-Orn”) in which β-alanine, ornithine and ornithine are peptide-bonded in this order from the N-terminal is effective as a protein stabilizer. Has already been proposed (patent document 1 unpublished at the time of filing this application). The outline is as follows.
[I] Reagent composition comprising a peptide having a peptide bond in the order of β-alanine and ornithine from the N-terminal, or ornithine and ornithine in the order of the N-terminal or a salt thereof, and at least one of a protein and a protein-binding carrier A method for stabilizing a protein, characterized by coexisting with a product.
[Ii] A protein stabilizer comprising a peptide having a peptide bond or a salt thereof in the order of β-alanine and ornithine from the N-terminal, or ornithine and ornithine from the N-terminal.
[Iii] A protein stabilizer comprising a dipeptide having a peptide bond in the order of β-alanine and ornithine, or a dipeptide having a peptide bond in the order of ornithine and ornithine or a salt thereof.
[Vi] Reagent composition comprising a peptide having a peptide bond in the order of β-alanine and ornithine from the N-terminus, or ornithine and ornithine in the order of N-terminus, or a salt thereof, and at least one of a protein and a protein-binding carrier A protein-containing solution characterized by coexisting with a product.
[V] A biological component measuring reagent comprising the protein-containing solution according to [vi].
[Vi] A standard solution used for measuring biological components, comprising the protein-containing solution according to [vi].

  Prior to this, the inventors of the present application have already proposed a method for producing the tripeptide from a swordfish extract (Patent Document 2). That is, it is a method of diluting a swordfish extract with water and fractionating. This document also discloses a chemical synthesis method using ornithine in which an α-amino group and a δ-amino group are protected and β-alanine in which a β-amino group is protected.

Japanese Patent Application No. 2005-239715 “Protein Stabilization Method, Protein Stabilizer, and Protein-Containing Solution” (Unpublished at the time of filing this application) JP-A 2005-200377 “Novel Tripeptide and Method for Producing the Same”

  In the research conducted by the inventors of the present application, the only natural raw material containing the tripeptide having the structure as described above is currently Shijimi. Therefore, when producing the tripeptide by separating and purifying it from a natural product, it is necessary to rely on 100% stigma as disclosed in Patent Document 2 above, and securing the stigma is essential. However, because the catch of the sea urchin varies depending on the natural environment and the price also changes, it is extremely unstable just to seek the raw material of the tripeptide directly from the sea urchin, and the most important stable supply as an industrial product is feared. .

  The tripeptide can also be chemically synthesized (Patent Document 2), but this requires expensive reagents and high technology. As an example, when the tripeptide was synthesized by a specialist in peptide synthesis, the cost was 135,000 yen at 25 mg. In other words, when commercializing the tripeptide as an industrial product, it should be avoided to rely on 100% swordfish, but it is expensive in chemical synthesis, so it is cheaper and more stable than chemical synthesis. There has been a demand for development of a method for producing a tripeptide.

The problem to be solved by the present invention, except for the above-mentioned problems of the prior art, is to produce a tripeptide having a specific structure (β-Ala-Orn-Orn) at a lower cost and more stably than chemical synthesis. It is to provide a tripeptide synthase solution and a method for producing the same, and to provide a method for synthesizing a tripeptide thereby.

As a result of studying the above problems, the inventors of the present application have added β-alanine and ornithine, which are constituent amino acids of the tripeptide, as substrates, added an enzyme solution prepared from a tissue of shijimi in the presence of ATP and Mg ²⁺ , and the tripeptide The present inventors have found a method for synthesizing and have arrived at the present invention based on this. That is, the invention claimed in the present application, or at least the disclosed invention, as means for solving the above-described problems is as follows.

(1) A method for producing an enzyme solution for synthesizing a tripeptide β-alanine-ornithine-ornithine using β-alanine and ornithine itself, both of which are not chemically modified, as a substrate. A tripeptide that removes at least the midgut gland so that digestion can be prevented or reduced, and uses a swordfish foot or scallop or both as a tissue to be extracted, and subjecting the tissue to an enzyme extraction step to obtain the enzyme solution Synthetic enzyme solution manufacturing method.
(2) The method for producing a tripeptide synthase solution according to (1), wherein the enzyme extraction step includes a separation operation or a concentration operation for leaving a fraction having a molecular weight of 100,000 or more.

The tripeptide synthase solution according to the present invention is not only a novel enzyme solution that has not been reported so far, but also has high industrial applicability. As a conventional peptide synthase, carnosine synthase (EC6.3.2.11), which is a synthase of carnosine (a dipeptide in which β-alanine and histidine are linked in the order of N-terminal), is known. There are no reports of industrial use. Carnosine is known for its functionality such as antioxidant effect, and a carnosine-enhanced extract prepared from chicken extract or the like is also commercially available. Although carnosine synthase is described to be extremely unstable and susceptible to inactivation (Reference 1), carnosine is universally present in muscles of mammals and fish, and carnosine is easily extracted. This is presumed to be the reason for the lack of synthase synthesis.

  In addition, an enzyme having dipeptide synthesis activity has been discovered from Bacillus subtilis (Kyowa Hakko Kogyo Co., Ltd.). This enzyme can directly bind amino acids and has succeeded in synthesizing various dipeptides including alanylglutamine. However, the synthesis of tripeptides by this enzyme has not been reported. Also, an enzyme that synthesizes a peptide by esterifying one type of amino acid and substituting this ester with the other amino acid has been discovered from microorganisms (Ajinomoto Co., Inc.). This enzyme is capable of synthesizing not only dipeptides but also further oligopeptides such as tripeptides.

However, in the synthesis using this enzyme, it is necessary to esterify the amino acid to be bound to the N-terminus. In contrast, the tripeptide synthase solution of the present invention does not require chemical modification such as esterification of amino acids at all, and uses β-alanine and ornithine, which are constituent amino acids of the tripeptide, as a substrate for simpler use. In addition, it is possible to produce a desired tripeptide at low cost.
(Reference 1: supervised by Maruo and Tamiya; Enzyme Handbook, p. 780 (1982), Asakura Shoten)

Since the tripeptide synthesis method, the tripeptide synthase solution, and the production method thereof according to the present invention are configured as described above, according to this, a specific structure (β-Ala) that is a useful component having a protein stabilizing effect is obtained. The tripeptide of -Orn-Orn) can be produced more inexpensively and stably without using chemical synthesis or direct extraction from natural products.

Hereinafter, the present invention will be described in more detail.
The tripeptide synthase solution of the present invention capable of synthesizing a tripeptide having a specific structure (β-Ala-Orn-Orn) can be extracted from one or a plurality of tissues constituting simimi meat. As will be described later, shijimi meat is composed of tissues such as mantle, gills, legs, scallops, midgut glands, gonads, etc. The enzyme solution extracted from only a part thereof is the present application. According to the present tripeptide synthase solution , a specific structure (β-Ala-Orn-Orn) is formed using β-alanine and ornithine itself, which are not subjected to chemical modification such as esterification shown in the above-mentioned prior art, as substrates. The tripeptide can be synthesized.

As a tissue used as a raw material for the tripeptide synthase solution , in particular, a swordfish leg, a scallop, or both can be used. In each tissue, there are many tripeptides of the above-mentioned specific structure, particularly in the legs and scallops. Therefore, it is considered that the tripeptide synthase is also contained in a large amount. On the other hand, the tissue to be specifically excluded from the raw material is the midgut gland. The activity of the protease contained in the midgut gland is considered to be high, and in order to prevent or reduce self-digestion by the protease, at least the meat part from which the midgut gland is removed is used as the tissue to be extracted.

As described in detail in Examples, the step of preparing an enzyme solution from rainbow trout meat tissue includes a separation operation or a concentration operation for leaving a fraction having a molecular weight of 100,000 or more. This is because the molecular weight of the target enzyme is estimated to be 100,000 or more.

As β-alanine, ornithine, ATP, and Mg ²⁺ used in the tripeptide synthesis method of the present invention, those generally marketed as reagents can be used. Ornithine is preferably a hydrochloride of L-ornithine, ATP is a crystalline product of adenosine 5′-triphosphate disodium trihydrate, and Mg ²⁺ is preferably magnesium chloride hexahydrate. But these were used.

In the preparation of the tripeptide synthase solution of the present invention, the tissue of shijimi is used as a raw material, but instead of homogenizing the whole meat and extracting the enzyme from it, as described above, in particular, the foot or scallop is cut off. And can be suitably used as a raw material. The inventors of this application classified the meat of Shijimi into the mantle, gill, foot, scallop, midgut and gonad tissues, and compared the tripeptide content in each tissue. As a result, it was found that a large amount of tripeptide was contained in the legs and scallops. Legs and shells with a high tripeptide content are considered to contain a large amount of tripeptide synthase, and these are used as particularly suitable raw materials.

  In addition, if homogenization is performed as a whole without particularly separating the tissue of the rainbow trout, there is a risk that the tripeptide synthase is digested by the action of the protease present in the tissue. In particular, the activity of proteases contained in the midgut gland is considered to be high, and therefore digestion by proteases can be reduced by separating the legs and scallops. In addition, legs are easier to separate between legs and shells.

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.
<Synthesis Method Using Tripeptide Synthetic Enzyme Solution of Tripeptide (Peptide Bonded in Order of β-Alanine, Ornithine, Ornithine, β-Ala-Orn-Orn) from N-terminal>
β-alanine aqueous solution (20 μmol / ml) 10 μl, ornithine aqueous solution (20 μmol / ml) 10 μl, MgCl ₂ aqueous solution (20 μmol / ml) 10 μl, ATP aqueous solution (20 μmol / ml) 10 μl, and 0.1 M potassium phosphate buffer (pH 7. 2) 50 μl was mixed, 10 μl of a tripeptide synthase solution prepared from the foot of <Example 4> described later was added and reacted at 25 ° C.

As β-alanine, ornithine, ATP, and Mg ²⁺ used in the tripeptide synthesis method, those commercially available as reagents are generally used. Ornithine is preferably L-ornithine hydrochloride, ATP is a crystalline product of adenosine 5′-triphosphate disodium trihydrate, and Mg ²⁺ is preferably magnesium chloride hexahydrate. These concentrations at the time of the synthesis reaction were set to 2 μmol / ml in the final reaction solution. A potassium phosphate buffer solution (pH 7.2) used as a buffer solution was prepared according to a conventional method, and was set to 50 mM in the final reaction solution. Although ATP is indispensable for the synthesis of the tripeptide, it was confirmed that ATP decreased with the generation of the tripeptide and conversely, AMP (adenosine 5′-monophosphate) was generated. In addition, although the density | concentration at the time of these reagents and reaction is recommended, this invention is not limited to these conditions.

The liquid was sampled from the reaction system 1 hour, 2 hours and 4 hours after the start of the reaction, and the state of tripeptide synthesis was examined by measuring the tripeptide. The tripeptide was measured using high performance liquid chromatography with reference to the method of Nagasawa et al. (Reference document 2). That is, the tripeptide was fluorescently labeled using orthophthalaldehyde and 2-mercaptoethanol, the column was Mightysil RP-18 GP (Kanto Chemical), and the mobile phase was 50 mM sodium acetate aqueous solution (pH 5.0): methanol: Detection was performed using a fluorescence detector (F-1050, Hitachi, Ex 340 nm, Em 455 nm) under the conditions of tetrahydrofuran = 35: 58: 7 and a flow rate of 1 ml / min.
(Reference 2: Nagasawa et al. Molecular and Cellular Biochemistry 225: 29-34, 2001.)

  FIG. 1 shows the results of high performance liquid chromatography showing the time course of tripeptide synthesis by the method of the present invention. As shown in the figure, it was confirmed that the target tripeptide was produced by the method of the present invention, and the tripeptide was found to increase over time.

When the tripeptide was synthesized with the above-mentioned mixed solution, the synthesis of the tripeptide was not observed when MgCl ₂ was not added. Similarly, synthesis of tripeptide was not observed when ATP was not added. Furthermore, no tripeptide synthesis was observed when using an enzyme solution heated in boiling water for 10 minutes. From this, it was found that the activity of the tripeptide synthase was lost by the heat treatment.

<Example 2 Preparation Method of Tripeptide Synthase Solution (1)>
70 fresh scallops (average weight 1.7 g / piece) were sanded for 3 hours at room temperature in running water. Thereafter, all 70 pieces were fried from water, and the whole meat including the midgut gland was taken out (yield 36.7 g). The collected tissue is cut into pieces using scissors, 450 ml of 20 mM potassium phosphate buffer (pH 7.2) is added, disrupted using a homogenizer, and centrifuged (10,000 × g, 30 minutes) using a centrifuge. )did. The supernatant fraction was desalted using a dialysis membrane having a fractional molecular weight of 10,000, and further concentrated to 15 ml using an ultrafiltration membrane having a fractional molecular weight of 10,000. The operation after sand removal was performed in an ice-cooled or cold room set at 4 ° C. The fluorescence intensity of the resulting solution used for the synthesis of the tripeptide <Example 1> as tripeptide synthetase solution was subjected to solution reacted for 4 hours at 25 ° C. in a high speed liquid chromatography, the synthetic tripeptide Was 207975.

<Example 3 Preparation Method of Tripeptide Synthase Solution (2)>
70 fresh scallops (average weight 1.7 g / piece) were sanded for 3 hours at room temperature in running water. Thereafter, all 70 pieces were fried from water and the shells were opened, and the tissue excluding the midgut gland was cut out from the whole meat (yield 14.2 g). The collected tissue is cut into pieces using scissors, 200 ml of 20 mM potassium phosphate buffer (pH 7.2) is added, disrupted using a homogenizer, and centrifuged (10,000 × g, 30 minutes) using a centrifuge. )did. The supernatant fraction was desalted using a dialysis membrane having a fractional molecular weight of 10,000, and further concentrated to 15 ml using an ultrafiltration membrane having a fractional molecular weight of 10,000. The operation after sand removal was performed in an ice-cooled or cold room set at 4 ° C. The fluorescence intensity of the resulting solution used for the synthesis of the tripeptide <Example 1> as tripeptide synthetase solution was subjected to solution reacted for 4 hours at 25 ° C. in a high speed liquid chromatography, the synthetic tripeptide Was 455513, and it was found that more tripeptides were synthesized than in <Example 2>.

<Example 4 Preparation Method of Tripeptide Synthase Solution (3)>
70 fresh scallops (average weight 1.7 g / piece) were sanded for 3 hours at room temperature in running water. Thereafter, all 70 pieces were fried from the water and the shells were opened, and only the legs or scallops were cut from the meat (yield: 1.75 g legs, 1.72 g scallops). Each collected foot or scallop is cut into pieces using scissors, added with 25 ml of 20 mM potassium phosphate buffer (pH 7.2), crushed using a homogenizer, and centrifuged (10,000 × g using a centrifuge). , 30 minutes). The supernatant fraction was desalted using a dialysis membrane having a fractional molecular weight of 10,000, and further concentrated to 15 ml using an ultrafiltration membrane having a fractional molecular weight of 10,000. The operation after sand removal was performed in an ice-cooled or cold room set at 4 ° C. Each solution obtained was used as a tripeptide synthase solution for the synthesis of the tripeptide of <Example 1>, and the solution reacted at 25 ° C. for 4 hours was subjected to high performance liquid chromatography. The fluorescence intensity was 961296 for the foot and 91621 for the scallop, and it was found that more tripeptides were synthesized than in Example 3 using either the foot or the scallop.

In this example, by operating under ice-cooling, it was possible to prepare a tripeptide synthase solution with retained activity from a separated foot without using a protease inhibitor. However, the use of a protease inhibitor such as PMSF (phenylmethanesulfonyl fluoride) is optional and does not interfere with this.

<Example 5 Preparation method of tripeptide synthase solution (4)>
The tripeptide synthase solution prepared from the foot according to Example 4 was fractionated using ultrafiltration membranes with a molecular weight cut off of 30,000, 50,000, and 100,000, and each fraction was used as an enzyme solution. Tripeptides were synthesized under the reaction conditions shown in Example 1> and analyzed by high performance liquid chromatography. As a result, when the permeated liquid in the ultrafiltration membrane having a molecular weight cut off of 30,000, 50,000 and 100,000 was used, that is, fractions having a molecular weight of 30,000 or less, 50,000 or less and 100,000 or less were obtained. When used as an enzyme solution , generation of tripeptide was not observed. When the residual liquid in the ultrafiltration membrane having a molecular weight cut off of 100,000, that is, a fraction having a molecular weight of 100,000 or more was used as the enzyme solution , generation of a tripeptide was observed. From this, it was found that the tripeptide synthase solution can be purified by adding a separation operation or a concentration operation to leave a fraction having a fractional molecular weight of 100,000 or more in the extraction step.

<Example 6: Properties of tripeptide synthase>
a. Molecular weight
The tripeptide synthase solution prepared from the foot according to <Example 4> was subjected to a Sephacryl S-300 gel filtration column (Amersham Bioscience, 2.3 × 118 cm). The eluate was 0.1M NaCl, flow rate 0.5ml / min, 4 ° C. 5 ml each was collected, and each fraction was used as an enzyme solution to try to synthesize a tripeptide in the same manner as in the method of <Example 1>, and the synthesized tripeptide was quantified by high performance liquid chromatography.

FIG. 2 is a chromatogram showing the results of gel filtration chromatography performed for calculating the molecular weight of the enzyme according to the tripeptide synthase solution of the present invention. As shown in the figure, Fraction No. The activity of tripeptide synthase was observed as a symmetrical peak at 45-53. From comparison with the elution position of a sample with a known molecular weight, Fraction No. which is the peak of enzyme activity. 49 was found to be an elution position having a molecular weight of about 190,000. That is, the molecular weight of the tripeptide synthase was estimated to be about 190,000.

b. Separation and purification by ion exchange column chromatography
The tripeptide synthase solution prepared from the foot according to <Example 4> was applied to a DEAE-Sephacel ion exchange column (Amersham Bioscience, 2 × 25 cm). The elution was performed using a 20 mM phosphate buffer (pH 7.2) with a linear concentration gradient with 0-0.5 M NaCl, and under conditions of a flow rate of 0.5 ml / min and 4 ° C. 5 ml each was collected, and each fraction was used as an enzyme solution to try to synthesize a tripeptide in the same manner as in the method of <Example 1>, and the synthesized tripeptide was quantified by high performance liquid chromatography.

  FIG. 3 is a chromatogram showing the results of ion exchange column chromatography performed to separate and purify the tripeptide synthase of the present invention. As shown in the figure, Fraction No. which is the position of 0.32M NaCl concentration. The activity of tripeptide synthase was observed as a symmetrical peak in 113-119. That is, Fraction No. It was found that the tripeptide synthase was purified at 113-119.

c. Optimal temperature In order to investigate the optimal temperature of the tripeptide synthase under the reaction conditions shown in <Example 1>, the reaction temperature was changed to 20 ° C. to 35 ° C. to try to synthesize the tripeptide. As a result, it was found that the optimum temperature of the tripeptide synthase under the above conditions was about 30 ° C.
FIG. 4 is a graph showing the optimum temperature measurement result of the tripeptide synthase of the present invention.

d. Substrate specificity In order to examine the substrate specificity of the tripeptide synthase under the reaction conditions shown in <Example 1>, L-alanine and γ-aminobutyric acid were substituted for β-alanine, and L-ornithine was substituted. A peptide synthesis was attempted using D-ornithine, L-histidine, L-lysine, and L-arginine as substrates (the reaction conditions other than the alternative amino acids were not changed), and detection was performed by high performance liquid chromatography. As a result, peptide synthesis was not observed for any of the alternative amino acids. Therefore, is this enzyme not a substrate for L-alanine other than β-alanine and L-ornithine, γ-aminobutyric acid, D-ornithine, L-histidine, L-lysine, and L-arginine under the present reaction conditions? Even if it became, the reaction rate was considered to be extremely slow compared with β-alanine and L-ornithine.

According to the present invention, it was confirmed that a tripeptide synthesized using a tripeptide synthase solution has a protein stabilizing effect described in Patent Document 1 described above. That is, chicken lysozyme (Sigma) was dissolved in 50 mM phosphate buffer (pH 7.1) to prepare a 1 mg / ml protein solution. To this solution, the tripeptide synthesized according to the present invention was added so as to have a concentration of 20 mM or 60 mM, followed by heat treatment at 98 ° C. for 10, 20 and 30 minutes. Next, 10 μl of the protein solution after heat treatment was added to 2 ml of 0.5 mg / ml Micrococcus lysodeikticus (Sigma) solution (100 mM phosphate buffer, pH 6.5), and a spectrophotometer (U-3310: Hitachi, Ltd.) The lysozyme activity was measured at an absorbance of 600 nm. As a result, 32.5% heating for 30 minutes was observed in the 20 mM addition section of tripeptide, and 80% residual activity was observed in the 30 mM heating section in the 60 mM addition section. According to the present invention, it was confirmed that a tripeptide synthesized using an enzyme solution has a high protein stabilizing effect.

  Patent Document 2 described above discloses a method for purifying the tripeptide from swordfish. When water is excluded, the swordfish extract contains about 50% of carbohydrates mainly composed of glycogen, about 35% of proteins, and about 10% of ash, and has a tripeptide content of about 1%. In order to purify the tripeptide containing only about 1% with high purity, after extracting the extract from shijimi, it is necessary to carry out processes such as ultrafiltration, gel filtration, ion exchange chromatography, etc. Is expected.

  In addition, Patent Document 2 shown above discloses a method for chemically synthesizing the tripeptide. From the viewpoint of ease of operation, it is preferable to synthesize by a solid phase method using a peptide synthesizer commercially available from each company, but special reagents and advanced techniques are required. That is, the peptide chain is sequentially extended from the C-terminal by the Fmoc method according to the program of the apparatus to be used. Ornithine [Fmoc-0rn (Boc)] in which the α-amino group and the δ-amino group were previously protected with Fmoc and Boc in order to use the carrier as a support for solid phase peptide synthesis and prevent side reactions. An Fmoc amino derivative of β-alanine [Fmoc-β-Ala] in which the β-amino group is protected with Fmoc is used. Fmoc-Orn (Boc) is bound to the carrier, and after deprotecting the α-amino group using piperidine or the like, the C-terminus of Fmoc-Orn (Boc) is coupled using a coupling agent, and piperidine is again used. To deprotect the α-amino group of the second ornithine and similarly couple the C-terminus of Fmoc-β-Ala. Finally, all of the deprotection and carrier removal are performed using an acid such as trifluoroacetic acid, and the tripeptide can be synthesized by freeze-drying the product extracted with 0.1% aqueous trifluoroacetic acid. For purification, a process such as reverse phase high performance liquid chromatography is added. Tripeptides can be chemically synthesized, but as is apparent from this description, special reagents, complicated operations, and high techniques are required.

This time, it has become possible to synthesize a tripeptide using an enzyme solution according to the present invention, but the tripeptide synthase solution is used in comparison with the case where a tripeptide is separated and purified from a swordfish extract, which is a mixture of various components . The tripeptide solution obtained by enzymatic synthesis was overwhelmingly free of impurities and can be easily purified. In addition, in the synthesis by enzyme, the peptides β-alanine and ornithine, and ATP and MgCl ₂ are dissolved in a reaction buffer, and the tripeptide is prepared by allowing a synthetic enzyme prepared from Shijimi to act on a mild condition of about 30 ° C. Compared with the chemical synthesis method, no special equipment such as a peptide synthesizer, amino acid with a protecting group or a special reagent is required, and high technology is not required. From these points, it is considered that the merit of synthesizing a tripeptide using an enzyme solution is great.

According to the tripeptide synthesis method, tripeptide synthase solution, and production method thereof of the present invention, a tripeptide having a specific structure (β-Ala-Orn-Orn), which is a useful component having a protein stabilizing effect, is chemically synthesized. It can be produced at a lower cost and more stably than chemical synthesis without being synthesized or directly extracted from natural products. Therefore, the invention has high industrial utility value.

It is a result of the high performance liquid chromatography which shows the time-dependent change of the tripeptide synthesis by the method of the present invention. It is a chromatogram which shows the result of the gel filtration chromatography performed in order to calculate the molecular weight of the enzyme which concerns on this invention tripeptide synthetase solution . It is a chromatogram which shows the result of the ion exchange column chromatography performed in order to isolate | separate and refine | purify this invention tripeptide synthetase. It is a graph which shows the measurement result of the optimal temperature of this invention tripeptide synthetase.

Claims (2)

A method for producing an enzyme solution used to synthesize the tripeptide β-alanine-ornithine-ornithine using β-alanine and ornithine itself, both of which are not chemically modified, as a substrate, which prevents self-digestion by protease from the meat of swordfish Alternatively, a tripeptide synthase solution that removes at least the midgut gland so that it can be reduced and uses the leg or scallop or both of them as a tissue to be extracted, and obtains the enzyme solution by subjecting the tissue to an enzyme extraction step. Production method. The method for producing a tripeptide synthase solution according to claim 1, wherein the enzyme extraction step includes a separation operation or a concentration operation for leaving a fraction having a molecular weight of 100,000 or more.

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