JP2560697B2 - Peptide assay - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for measuring a peptide, and more specifically, a method for measuring an enzyme electrode capable of quickly and accurately measuring a peptide produced by hydrolysis of a protein by a simple operation. It is about.

[Prior Art] In recent years, in the field of food processing technology, various proteins such as soybean protein, milk protein, blood protein, and fish protein are hydrolyzed by proteolytic enzymes (proteases), and so-called functional proteins with higher utility value. Various techniques have been tried. In such a case, a technique for measuring the peptide produced by the decomposition is required as a method for knowing the degree of protein decomposition. Further, such a technique is also useful for knowing the maturity of brewed foods such as soy sauce and miso.

The following methods are known as methods for measuring the hydrolysis rate of a raw material protein from the content of amino acids and peptides produced by the decomposition of proteins as indexes.

(A) A method in which water-soluble nitrogen and water-insoluble nitrogen in a protein are measured by the Kjeldahl method, and the ratio of the water-soluble nitrogen content to the total nitrogen content is used as the proteolysis rate.

(B) A method of estimating the proteolysis rate by measuring the protein concentration in the water-soluble portion by the Folin method.

(C) The protein concentration in the water-soluble portion was determined to be 280 nm or 214
A method of estimating the proteolysis rate by measuring from the UV absorbance at nm.

(D) A method of estimating the proteolysis rate by obtaining the protein concentration of the water-soluble portion from the absorption peak of the peptide in a fixed fraction by chromatographic analysis.

The above methods (a) to (d) are all methods for estimating the rate of proteolysis with respect to the total amount of protein by measuring the concentration of a relatively low molecular weight water-soluble protein.

Alternatively, in addition to the above-described methods (a) to (d), an electrophoretic method for measuring a specific protein, peptide, amino acid or the like is also known as a method utilizing electric properties.

[Problems to be Solved by the Invention] However, all of the above-mentioned methods have a drawback that the analysis operation is complicated and that it takes a lot of time to estimate the proteolysis rate. That is, with these methods, it is extremely difficult to feed back the measurement results to the food manufacturing process and use them for product quality control.

The present invention has been made in view of such circumstances, and an object thereof is to provide a method for rapidly and accurately measuring the amount of peptide by a simple operation.

[Means for Solving Problems] According to the present invention capable of achieving the above object, L-amino acid oxidase is allowed to act on an amino acid mixture produced by allowing leucine aminopeptidase to act on a protein hydrolyzate,
The gist is to detect a redox current or a potential change corresponding to hydrogen peroxide generated as a result of an enzymatic reaction.

[Operation] The present invention is configured as described above, but the point is that the configuration in which the redox current or the potential change corresponding to hydrogen peroxide generated as a result of a series of enzymatic reactions is detected is employed to determine the peptide abundance in an electrical manner. Since the amount of change is detected as the amount of change, the amount of peptide and thus the rate of proteolysis can be measured quickly and accurately.

 The principle of the method of the present invention will be described step by step.

The protein of the present invention is partially hydrolyzed by a proteolytic enzyme, which is apparent from the above-mentioned meaning. In this protein (hydrolyzate),
It includes those decomposed into amino acids, those decomposed into a peptide state, and the like. This is because the above-mentioned proteolytic enzyme has the specificity of rapidly cleaving only the specific bond of the protein.

The present invention measures the amount of peptides contained in a protein of interest. Although the target protein sample contains amino acids in addition to the peptides as described above, the amount of this amino acid is generally a negligible amount. Therefore, the peptide amount can be measured by a series of operations described below, but in some cases, it may contain a large amount of amino acids that cannot be ignored. That is, the measurement of the amount of peptide is obtained from the amount of amino acid generated by the decomposition of the peptide as described later, and when a large amount of amino acids are contained from the beginning, the measurement of the peptide is affected. In such a case, it is necessary to simultaneously measure and correct the amount of amino acid contained from the beginning by the method described later.

When leucine acinopeptidase is allowed to act on a protein hydrolyzate, the peptides contained in the hydrolyzate are decomposed into amino acids by the action of leucine aminopeptidase. Most of the released amino acids are L because they are produced by the decomposition of naturally occurring proteins.
Type amino acid. That is, there are both L-type and D-type amino acids existing in the living body, but most of the amino acids existing in natural proteins are L-type amino acids.
The liberated L-amino acid is further decomposed by the action of L-amino acid oxidase to generate hydrogen peroxide (H ₂ O ₂ ) proportional to the peptide concentration. The reaction mechanism at this time is represented by the following equation (1).

When H ₂ O ₂ produced by the above reaction is reduced by a polarographic electrode, an electrode current (oxidation-reduction current) proportional to the H ₂ O ₂ concentration is generated. Then, measure the electrode current of a peptide standard solution of known concentration to create a calibration curve in advance, and measure the amount of peptide contained in the target protein by comparing this calibration curve with the obtained electrode current. You can

In order to achieve the effect of the present invention more effectively, the following two embodiments can be proposed. That is,
(A) using an immobilized enzyme immobilized on a carrier as leucine aminopeptidase and L-amino acid oxidase; and (B) having leucine amino in the vicinity of the first electrode, which has first and second electrodes. For example, a peptidase and an L-amino acid oxidase-immobilized enzyme are arranged, and an L-amino acid oxidase-immobilized enzyme is arranged in the vicinity of the second electrode to detect a redox current or a potential change.

The effect of the above (A) is that by using two kinds of enzymes to be used as immobilized enzymes, the peptides can be easily and accurately and in a short time without being affected by the colored substances and coexisting substances contained in the target protein. Can be measured and stable measurement can be performed for a long time.

The effect of (B) is particularly effective when only the peptide in the protein containing a large amount of L-amino acid is measured from the beginning. That is, a series of enzymatic reactions according to the present invention is made to proceed by disposing a leucine aminopeptidase- and L-amino acid oxidase-immobilized enzyme in the vicinity of the first electrode, and at the same time, in the vicinity of the second electrode, L-
By arranging an immobilized enzyme of amino acid oxidase, the amino acid contained from the beginning is measured and corrected by the difference between the two. By making such a correction, the amount of peptide can be accurately measured without being affected by the L-amino acid initially contained in the target protein.

In the present invention, leucine aminopeptidase and L-amino acid oxidase are used, and the origin is not particularly limited, or microorganisms or organelles (cellular organelles) that produce these enzymes are used. Is also good.

For the immobilization of enzymes (or microorganisms etc.), all commonly known methods can be adopted without any limitation.
For example, in a chemical bonding method such as a crosslinking method or a covalent bonding method, an enzyme can be immobilized on a functional group such as an amino group, a hydroxyl group, or a carboxyl group contained in an enzyme or a carrier via a crosslinking agent such as glutaraldehyde or hexamethylene diisocyanate. . The enzyme can also be immobilized by using a diazo coupling method, a cyanogen bromide activation method, a triazinyl derivative method, a halogenoacetyl derivative method, an acid azide derivative method, or a carboxychloride derivative method. Furthermore, in the inclusion method, synthetic polymers such as polyacrylamide, polyvinyl alcohol, polyhydroxyethyl acrylate, or various photocurable photosensitive resins or natural polymers such as chitosan, gelatin, and carrageenan are used as the inclusion matrix, and enzymes and the like are used. It can be fixed.

Considering the shape of the immobilized enzyme, considering (a) that it can be integrated when constructing the enzyme electrode, and (b) that only the substrate and the electrode detection substance can be transmitted without transmitting the interfering substance, etc. Optimally, it is the shape. However, in some cases, a fibrous or granular carrier may be used and used as a bioreactor type column, which may be appropriately determined.

Examples of the electrode used in the present invention include platinum (Pt)
Examples thereof include a polarographic electrode composed of an anode and a silver (Ag) cathode, a carbon dioxide gas electrode and an ammonium ion electrode. Also, the configuration of the actual current measuring device is not limited in any way, and a reactor system in which the reaction of the immobilized enzyme is performed using a column and then the sensing substance is electrically measured by an electrode, or the immobilized enzyme is a membrane Examples of the shape include an enzyme electrode system integrated with an electrode.

[Examples] The present invention will be described in more detail with reference to Examples below.

Example 1 A porous layer of a 4.5 μm-thick acetylcellulose-based asymmetric structure film was prepared by mixing 0.1 μm of γ-aminopropyltriethoxysilane.
It was immersed in a solution consisting of 1 ml of acetic acid, 0.03 ml of acetic acid and 1 ml of distilled water, air-dried at room temperature for 30 minutes, and then heat-treated at 95 ° C. for 60 minutes. Then 0.
It was immersed in a 1N sodium hydroxide solution for 10 minutes and thoroughly washed with distilled water.

The obtained aminated acetyl cellulose membrane was immersed in a 5% glutaraldehyde aqueous solution and treated at room temperature for 10 minutes to introduce aldehyde groups on the membrane surface. On the porous surface 60 cm ² of the acetyl cellulose asymmetric structure membrane introduced with an aldehyde group,
Leucine aminopeptidase (EC.3.4.11.2,8.4IU / mg)
5 mg, L-amino acid oxidase (EC.1.4.3.2.0.13IU / m
g) 5 mg, bovine serum albumin 20 mg, 0.05 M phosphate buffer (pH
7.0) 150μ and 4% glutaraldehyde aqueous solution 50μ
The enzyme solution consisting of was uniformly cast and reacted at 4 ° C. for 30 minutes.

Then, the reaction was stopped with a 1 M glycine solution, and then washing with 0.05 M phosphate buffer (pH 7.0) was sufficiently repeated. Then, in order to prevent the immobilized enzyme from being contaminated, a porous polycarbonate membrane having a pore size of 0.05 μm was laminated and then air-dried at 4 ° C. to obtain an immobilized enzyme membrane I.

When the activities of leucine aminopeptidase and L-amino acid oxidase in the obtained immobilized enzyme membrane I were examined, when L-leucyl-p-diethylaminoanilide and L-leucine were used as substrates, 0.24 IU / cm ² , respectively.
It was 0.016 IU / cm ² .

In the same manner as the procedure for obtaining the immobilized enzyme membrane I, L
-Immobilized enzyme membrane I (0.034I
U / cm ² ) was obtained.

Next, a hydrogen peroxide detection type polarographic electrode consisting of Pt-anode and Ag-cathode was prepared, and the immobilized enzyme membrane I (hereinafter referred to as a peptide sensor) was provided in the vicinity of the first electrode.
The immobilized enzyme membrane II (hereinafter referred to as an amino acid sensor) was placed in the vicinity of the second electrode.

Furthermore, each of the above sensors was attached to a flow system analyzer, and L-leucyl was used under the conditions of using 0.05M phosphate buffer (pH 7.0, 37 ° C), buffer flow rate 2.0 ml / min, and sample volume 10μ. The responsiveness to glycylglycine and L-leucine was investigated.

The response curve of the peptide sensor to L-leucylglycylglycine is shown in FIG. 1, and the response curve of the amino acid sensor to L-leucine is shown in FIG. The first
In the portions shown by (1) to (6) in Fig. 2, the concentrations are (1) 0.1, (2) 0.2, (3) 0.3, (4) 0.4, (5) 0.
5, (6) 1.0 (millimol /).
As is clear from the results of FIGS. 1 and 2, it is understood that each substrate concentration has a good linear relationship with the sensor output in the range of 0 to 1.0 mM.

Further, in Table 1 below, the outputs of the respective sensors for dipeptides and oligopeptides are shown as relative outputs for L-leucine. In Table 1, Leu, Gly, Phe and Tyr
Represent leucine, glycine, phenylalanine and tyrosine, respectively.

The results in Table 1 show that the amino acid sensor does not respond to amino acids other than amino acids, and that the peptide sensor reacts not only with amino acids such as L-leucine but also with oligopeptides such as dipeptides and tripeptides. There is. This shows that the method of the present invention gives accurate results for the measurement of peptides.

Example 2 5 g of milk casein was dissolved in 35 ml of 0.1N phosphoric acid solution, and the mixture was mixed at 80 ° C. for 10 hours.
Heated for minutes. After cooling to room temperature, adjust the pH to 7.0 with 0.1N sodium hydroxide and add distilled water to bring the total volume to 100 ml.
And Protease-CE (manufactured by Amano Pharmaceutical Co., Ltd.) was added to 4 parts of the 5% casein solution adjusted in this way at 0.31.
A part of each enzyme solution was added so as to be 3,62.5,125,250 U / dl and reacted at 40 ° C for 30 minutes to hydrolyze casein. After the reaction, the protease was inactivated by heating at 80 ° C. for 10 minutes. After acetic acid was added to the hydrolyzed casein solution for acid precipitation, the N content of soluble protein in the supernatant was measured by the Kjeldahl method, and the ratio of casein to the total N content was defined as the proteolysis rate. The decomposition rate obtained at this time was controlled respectively,
It was 12.3, 21.4, 34.8 and 46.8%.

With respect to the above-mentioned 5 kinds of hydrolyzed casein solutions, the response curves by the peptide sensor shown in Example 1 were investigated. The result is shown in FIG. The results shown in FIG. 3 are obtained by using L-leucylglycylglycine or L-leucine as a standard solution, and (1) in the figure is 0.5 mmol / mol.
Shows the response curve of the L-leucylglycylglycine standard solution in (2) to (6) and shows the response curve of the casein solution having each of the above decomposition rates.

When the relationship between the apparent concentration of the peptide obtained in FIG. 3 and the decomposition rate was investigated, the results shown in FIG. 4 were obtained.

As is clear from the results shown in FIG. 4, the amount of peptide in casein significantly increases with the decomposition rate, and a good proportional relationship is observed between the two. Therefore, the method for measuring a peptide according to the method of the present invention can measure the rate of protein degradation with an accuracy comparable to the Kjeldahl method, and is a very useful technique as a method for measuring a peptide.

Furthermore, the present inventors also investigated the measurement durability of each sensor according to the present invention. That is, the initial output of each sensor for L-leucylglycylglycine and L-leucine was set to 100%, and changes in relative output (%) depending on the elapsed days were investigated. The conditions are 0.05M phosphate buffer, pH 7.0, 37
Performed at ° C. The results are shown in FIG. 5, and 105% (peptide sensor) and 141% (amino acid sensor) were retained even after 40 days had elapsed.

[Advantages of the Invention] As described above, according to the present invention, by adopting the described configuration, the amount of peptide can be measured quickly and accurately by a simple operation.

[Brief description of drawings]

Fig. 1 shows the response curve of the peptide sensor to L-leucylglycylglycine, and Fig. 2 shows the amino acid sensor of L
-Leucine response curve, Fig. 3 is the response curve of the peptide sensor to various casein solutions used in Example 2, and Fig. 4 is the concentration measured by the method of the present invention and the Kjeldahl decomposition rate for peptides and amino acids in the casein solution. FIG. 5 is a graph showing the relationship with the above, and FIG. 5 is a graph showing the change in relative output (%) depending on the number of days elapsed in each sensor.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location G01N 27/30 353U

Claims (3)

(57) [Claims] 1. A redox current or potential change corresponding to hydrogen peroxide produced as a result of oxygen reaction is detected by allowing L-amino acid oxidase to act on an amino acid mixture produced by allowing leucine aminopeptidase to act on a protein hydrolyzate. A method for measuring a peptide, which comprises: 2. The leucine aminopeptidase and L-
The method according to claim 1, wherein an immobilized enzyme immobilized on a carrier is used as the amino acid oxidase. 3. A first electrode and a second electrode, wherein a leucine aminopeptidase and an immobilized enzyme of L-amino acid oxidase are arranged in the vicinity of the first electrode, and an L-amino acid is arranged in the vicinity of the second electrode. The assay method according to claim 1 or 2, wherein an oxidase-immobilized enzyme is arranged to detect a redox current or a potential change.