KR890003087B1 - L-glutamin acid of analysis use kit and biosensor - Google Patents

Abstract

The biosensor and analysis kit of L-glutamic acid are presented. Thus L-glutamic acid oxydase having strong affinity and high specificity is prepared with an analysis reagent containing the above enzyme for analysis with the kit.

Description

L-Glutamic Acid Analysis Kits and Biosensors

1 is a graph showing the pH range of action of the enzyme (solid line) and known enzyme (dotted line) of the present invention.

Figure 2 is a graph showing the stable pH range (37 ℃, 60 minutes) of the enzyme (solid line) and known enzyme (dotted line) of the present invention.

3 is a graph showing a stable pH range of the enzyme of the present invention (45 ℃, maintained for 15 minutes).

4 is a graph showing a stable pH range of the enzyme of the present invention (60 ℃, maintained for 15 minutes).

5 is a graph showing the functional temperature range of the enzyme of the present invention.

Figure 6 is a graph showing the stable temperature range of the enzyme (solid line) and known enzyme (dotted line) of the present invention.

7 is a graph showing the ultraviolet absorption spectrum of the enzyme of the present invention.

8 and 9 are graphs respectively showing calibration curves of L-glutamic acid in the case of using the phenol solution or the dimethylaniline solution as the reagent B in the kit of Example B1.

10 is a graph showing an analytical curve of L-glutamic acid in Example B2.

11 is a graph showing a calibration curve of L-glutamic acid in Example B3.

12 is a graph showing a calibration curve of GOT activity in Example B4.

FIG. 13 is a graph showing a calibration curve of GPT activity in Example B4. FIG.

14 is a graph showing an analytical curve of L-glutamic acid in Example B5.

15 is a graph showing an analytical curve of L-glutamic acid obtained using the enzyme electrode of Example B6.

Fig. 16 is a graph showing the calibration curve of glutaminase activity in Example B7.

17 is a schematic view showing an L-glutime acid sensor according to the present invention.

FIG. 18 is a schematic view showing an L-glutamic acid analyzer using the L-glutamic acid sensor of FIG. 17. FIG.

FIG. 19 is a graph showing the relationship between sodium L-glutamate concentration and current reduction value when using the apparatus of FIG. 18. FIG.

FIG. 20 is a graph showing the responsiveness at each temperature when using the apparatus of FIG. 18. FIG.

FIG. 21 is a graph showing the responsiveness at each pH when using the apparatus of FIG. 18. FIG.

22 is a graph showing the relationship between the L-glutamic acid concentration and the current value when the L-glutamic acid analyzer of Example C2 is used.

FIG. 23 is a graph showing the relationship between the addition amount and recovery amount of sodium L-glutamate in the apparatus of FIG.

24 is a graph showing a graph of the addition amount and recovery amount of sodium L-glutamate when the L-glutamic acid analyzer of Example C2 is used.

The present invention relates to kits and biosensors for the analysis of the novel enzyme L-glutamic acid.

More specifically, the present invention has a strong affinity for L-glutamic acid and a high substrate specificity, substantially hardly acts on other amino acids, and is highly stable L-glutamic acid oxidase, a method for producing by the microorganism, Analysis of L-glutamic acid in an assay sample using this enzyme, reagent for analyzing L-glutamic acid containing this enzyme, kit for analysis of L-glutamic acid using this enzyme, and biosensor using the enzyme ).

Conventionally, chromatographic methods, microbial quantification methods, electrophoresis methods, and enzyme methods are known as L-glutamic acid analysis methods, but chromatographic methods and enzyme methods are generally used.

As a chromatographic method, a method using an amino acid automatic analyzer is common. This method has high precision and reliability and is an excellent method. However, the apparatus is expensive, the protein needs to be removed depending on the sample, and the sample preparation method is complicated.

As the enzyme method, (1) a method using L-glutamic acid decarboxylase and (2) a method using L-glutamic acid dehydrogenase are known. However, the method of using these known enzymes has the following problems.

(1) In the method of using L-glutamic acid decarboxylase, the reaction is measured by detecting carbon dioxide gas, which is a reaction product, and (a) using a Warburg pressure gauge. The method or (b) the method of using an automatic analyzer is adopted.

The method of using the Warbruck pressure gauge of (A) is high in accuracy but requires considerable skill, requires a long time for measurement, and has a low sample processing capacity. In addition, the method of using the automatic analyzer of (b) is a method of absorbing carbon dioxide gas into sodium carbonate solution of phenolphthalein and measuring the amount of carbon dioxide generated at darkness. There is a problem in that preliminary operation is required, such as cooling while degassing oxygen, gas-liquid separation is required after the reaction, and the apparatus is complicated.

As glutamate decarboxylase, an enzyme derived from pumpkin or Escherichia coli is generally used, but an enzyme derived from pumpkin has urease activity, and when a sample containing urea is used, There is a concern that measurement errors may occur, and since this enzyme is inhibited by organic acids such as acetic acid, there is a drawback that the enzyme activity is inhibited in a sample containing a large amount of organic acids and thus an accurate result is not obtained. In addition, since E. coli-derived enzymes exhibit activity against L-arginine and L-glutamine, accurate results are not obtained when a sample containing a large amount of these amino acids is used. In addition, the retention rate of the enzyme itself is also poor.

(2) L-glutamic acid dehydrogenase reacts L-glutamic acid in the presence of NAD (oxidized nicotinamide adeninedinucleotide) to produce α-ketoglutaric acid, ammonia and NADH (cyclized nicotinamide adenine dinucleotide) Although the reaction is catalyzed, in the method using this enzyme, the reaction is measured by detecting an increase in absorbance at 340 nm for the amount of NADH produced. However, the parallelism of this enzymatic reaction is inclined to L-glutamic acid formation, and in order to analyze L-glutamic acid using this enzyme, the equilibrium of the reaction must be inclined toward producing α-ketoglutaric acid. Therefore, various studies are required. Although the trapping agent of (alpha)-ketoglutaric acid is normally added to the reaction system for this purpose, reaction may be inhibited unless the concentration is adjusted strictly. In addition, the NAD concentration must also be strictly controlled. In addition, when the sample containing the substance which shows absorption in measurement wavelengths, such as soy sauce, is used, the value obtained by reaction must be corrected using the value of a blank test. In addition, lactic acid dehydrogenase activity may exist in the enzyme to be used, so the influence of such enzyme activity must also be considered.

Recently, L-amino acid oxidase having high substrate specificity to L-glutamic acid belongs to a microorganism belonging to the genus Streptomyces (hereinafter sometimes referred to as "S"), specifically, Streptomyces bio license (Streptomyces). violascens) was found to be produced (see Japanese Patent Application Laid-Open No. 57 (1982) -43685). Although the physicochemical properties of this glutamic acid oxidase (sometimes abbreviated as "public enzyme" below) as proteins are not certain, the properties described as enzymatic properties are as follows.

(1) substrate specificity

When the activity against L-glutamic acid is set to 100, it shows a relative activity of 8, 4 for L-glutamine, 6.8 for L-histidine, and no substantial activity for other amino acids.

(2) optimum pH

ph 5-6

(3) pH stability

It is stable in the range of pH 3.5-6.5 (37 degreeC, hold for 1 hour).

(4) temperature stability

Stable to 50 ° C (hold for 10 minutes).

(5) effect of inhibitor

Almost completely inhibited by mercury ions, copper ions and diethyldithio carbamate.

There are various problems in order to use such known enzymes for the analysis of L-glutamic acid.

Known enzymes have a higher substrate specificity for L-glutamic acid than conventionally known L-amino acid oxidases, but are apparently active against other amino acids as described above. Specific quantification of L-glutamic acid in the presence of these amino acids It cannot be used for the characterization. In addition, known enzymes do not have high safety, and it is not considered that the preservation safety and the use stability as reagents for analysis are also good. In the case where copper ions are present in the sample to be analyzed, known enzyme activity is markedly inhibited, which makes it difficult to analyze. In addition, the pH of the reaction solution used in various clinical biochemical assays, particularly in the analysis of enzymatic activity in the blood, is usually neutral, and the known enzymes lose their activity completely at pH 7.5 when treated at 37 ° C. for one hour. It is difficult to use for the well-known enzyme analysis in pH range.

In addition, although a conventional method for analyzing L-amino acids using L-amino acid oxidase has been known, known L-amino acid oxidase is difficult to act on L-glutamic acid, and a specific method of L-glutamic acid may be used by such an enzyme. The analysis could not be done.

On the other hand, a method of using a biosensor as another analysis method of L-glutamic acid is known.

Conventionally, as a biosensor for analyzing L-glutamic acid, (1) an enzyme electrode using L-glutamic acid dehydrogenase as a receptor of L-glutamic acid and using a cationic electrode as a transregulator part (Anal.Chim.Acta, 56) , 333 (1971)), and (2) using the freeze-dried gyunje of Escherichia coli (E.coli) represents the L- glutamic acid decarboxylase enzyme activity as LES acceptor portion of L- glutamic acid and a carbon dioxide electrode as the transducer West The microbial electrode used (Anal. Chim. Acta, 116, 61 (1980)) is known.

The enzyme electrode of (1) is extremely poor in enzyme safety (2 days), and since a cationic electrode is used, it is easy to receive a positive effect of a cation in which sodium ions, potassium ions and the like coexist, and are not practical. The microbial electrode of (2) uses immobilized microbial cells, which is excellent in stability (more than 1,500 times for 3 weeks), but under aerobic conditions, carbon dioxide gas is generated by the respiratory action of the cells and affects the measurement. In order to remove such effects, it is necessary to inhibit the respiratory action of the cells by injecting nitrogen gas and to remove carbon dioxide gas from the sample. In addition, since the microorganism electrode does not have high substrate specificity, it is considered that it can be used only for the rough measurement such as the process control of fermentation.

Substrate Specificity of Microbial Electrode

*: Use of acetone treated cells

An enzyme electrode using L-amino acid oxidase as the receptor portion is also known (Anal. Chem. 47, 1359 (1975)). However, the conventional L-amino acid oxidase used for such an enzyme electrode hardly acts on L-glutamic acid, and the specific analysis of L-glutamic acid was not possible even if the enzyme electrode was used.

Whether or not such a known enzyme can be used in a biosensor for specifically analyzing L-glutamic acid is not disclosed in the Japanese Laid-Open Patent Publication, but is known in the receptor portion of the biosensor, for example. Even if the enzyme is used, various problems are considered. In other words, the known enzyme has a high substrate specificity for L-glutamic acid compared to the known L-amino acid oxidase, but still exhibits a clear activity to other amino acids as described above, and the specificity of L-glutamic acid in the coexistence of these amino acids. It cannot be used as a biosensor for performing analytical analysis. In addition, known enzymes do not have high stability, and even when used in the receptor portion of a biosensor, storage stability and use safety are not necessarily good. In addition, since the enzyme is extremely unstable at pH 7 or higher, it is sometimes difficult to use the biosensor of the known enzyme at neutral pH where various biochemical analyses are performed in the clinical field. In the case where copper ions are present in the sample to be analyzed, it is considered that the known enzyme is significantly inhibited and the analysis becomes difficult.

As described above, there is no biosensor for L-glutamic acid analysis using an oxidase as a receptor portion for specifically identifying L-glutamic acid, and there is no L-amino acid oxidase which can achieve the above object. Did not.

The inventors of the present invention searched for an enzyme that oxidatively deaminoizes L-amino acid with respect to a culture of microorganisms. As a result, L-glutamic acid has a very high substrate property in the culture of actinomycetes newly isolated from nature. -Amino acid oxidase was found to exist, and the enzyme of the present invention was purified and isolated as a single enzyme protein in a culture of such microorganisms to complete the present invention.

The present invention has the action of producing an α-ketoglutaric acid, ammonia and hydrogen peroxide by oxidatively deaminoating the α-amino group of L-glutamic acid in the presence of water and oxygen, and having a very high substrate specificity for L-glutamic acid. It is to provide L-glutamic acid oxidase which is a highly stable L-amino acid oxidase which does not act substantially at all on L-glutamine and L-histidine.

The present invention is characterized in that the microorganism belonging to the genus Straptomyses, the microorganism having the L- glutamic acid oxidase production capacity is cultured in a medium in which the microorganism can be grown, and the L-glutamic acid oxidase is collected from the culture. It is to provide a method for producing L-glutamic acid oxidase.

The effect of the present invention acts specifically on L-glutamic acid and does not substantially act on other amino acids, so it is suitable for quantification of L-glutamic acid in a system containing many kinds of amino acids. For example, it can be used for process control or process analysis or screening of glutamic acid-producing bacteria in the fields of measurement of glutamic acid content in foods such as soy sauce, ekisu, liquid seasoning, glutamic acid fermentation or soy sauce production. In addition, enzymes such as glutamic acid, glutamic acid-oxaloacetic acid transaminase (GOT), glutamic acid-pyruvic acid transaminase (GPT), and glutamic acid transpeptidase (X-GTP) are used as products of glutamic acid. Since activity measurement can be easily performed using the enzyme of the present invention, it is also useful in the field of clinical examination or biochemistry.

In addition, since the enzyme of the present invention is an oxidase reaction which is most widely used in clinical tests or food analysis, the enzyme reaction can also be used as it is. There is an advantage to that.

In addition, the enzyme of the present invention has high stability even when compared with a general enzyme for analysis, including a known enzyme, and thus can be used as an enzyme electrode for glutamic acid sensor, and can be used as a labeling enzyme for measuring enzyme immunity (Japanese Patent Application Laid-Open No. 57 (1982). ) -37261) is also expected, and the analytical reagent of the present invention is stable in storage and use, and has high versatility and economy.

In another aspect, the present invention is applied to L- glutamic acid oxidase and L- glutamic acid in the presence of oxygen and water, and to detect the consumption of oxygen or the production of hydrogen peroxide, ammonia or α-ketoglutaric acid according to the reaction To provide an analysis of glutamic acid.

In addition, the present invention relates to the L-glutamic acid oxidase for L-glutamic acid in an assay sample containing a large amount of L-aspartic acid together with L-glutamic acid to substantially affect the analysis of the L-glutamic acid of the present invention. When reacted in the presence of oxygen and water, it reacts at pH 5-6, and provides an assay for L-glutamic acid, characterized by detecting the consumption of oxygen or the production of hydrogen peroxide, ammonia or α-ketoglutaric acid according to the reaction. It is.

The present invention also provides a reagent for analyzing L-glutamic acid, which comprises the L-glutamic acid oxidase.

The present invention also provides a kit for the analysis of L- glutamic acid consisting of the L- glutamic acid oxidase and the reagent for detecting the reaction by the enzyme.

The present invention also provides a biosensor comprising an enzyme serving as a receptor portion of a substance to be detected and a transducer portion for detecting chemical or physical changes in an analytical sample caused by the action of the enzyme and converting the same into an electrical signal. It is to provide a biosensor characterized by using L-glutamic acid oxidase having a very high substrate specificity to glutamic acid, substantially not acting on amino acids other than L-glutamic acid, and having high stability.

The biosensor of the present invention is different from known biosensors for L-glutamic acid analysis, and since the oxidase which is most widely used as the receptor portion of the biosensor is used, its production is easy. The means for detecting the enzyme reaction can be selected according to the purpose from a known means such as a means for detecting a decrease in oxygen as a substrate or a means for detecting an increase in hydrogen peroxide or ammonia as a reaction product. In addition, the biosensor of the present invention has a particularly high specificity for L-glutamic acid as compared with known biosensors, and even a sample containing multiple amino acids can selectively analyze only L-glutamic acid without interfering with other amino acids. .

As described above, by using the biosensor of the present invention, not only L-glutamic acid in an analytical sample containing L-glutamic acid as its original component such as food and fermentation broth can be selectively analyzed, but also a reaction system involving L-glutamic acid. For example, glutaminase, glutamic acid racease, glutamic acid-oxaloacetic acid transaminase (GOT), glutamic acid-pyruvic acid transaminase (GPT), Υ-glutamyl transpeptidase (VII-GPT) For example, the enzyme activity in the reaction system of an enzyme involving L-glutamic acid or another substance involved in the reaction can be analyzed.

(I) the enzyme of the present invention

The enzyme of the present invention has a very high substrate specificity to L-glutamic acid, does not substantially act on amino acids other than L-glutamic acid, and may be a highly safe L-glutamic acid oxidase, regardless of its origin or origin, and regardless of its production method. Attach to

Next, enzymes obtained by culturing microorganisms belonging to Streptomyces are taken, and specific properties and production methods are described for this exemplary enzyme.

(A) Enzymatic and Physicochemical Properties of the Invention

The enzymatic and physicochemical properties of the purified enzyme product of L-glutamic acid oxidase prepared by the method of Example A1 described below are as follows.

(1) action

When the enzyme of the present invention uses L-glutamic acid as a substrate, 1 mole of oxygen and 1 mole of water per 1 mole of L-glutamic acid, 1 mole of α-ketoglutaric acid, 1 mole, Produces ammonia and 1 mole of hydrogen peroxide.

(2) substrate specificity

The result of having made the purified product of the enzyme of this invention act on various amino acids is a 1st table | surface. The concentration of each substrate was 10 mM, and the reaction was performed at pH 7.4 (0.1 M potassium phosphate buffer) and pH 6.0 (0.1 M acetic acid buffer). The enzyme activity was measured by the oxygen electrode method described below and expressed as a relative value of the activity with respect to L-glutamic acid.

[Table 1]

As described above, the enzyme of the present invention has high substrate properties for L-glutamic acid, and shows only a slight activity (0.6%) to L-aspartic acid at pH 7.4 for other amino acids, and contains L-glutamic acid and L-histidine. There is practically no activity with other L-amino acids and D-glutamic acid, and practically no activity with L-aspartic acid at pH 6.0.

With respect to the enzyme of the present invention, the above-mentioned known enzymes do not show activity against L-aspartic acid (0.1% or less), but are enzymes showing activity of 8.4% for L-glutamine and 6.8% for L-histidine, respectively. However, they differ in substrate specificity. The Km value for L-glutamic acid of the enzyme of the present invention is 2.1 × 10 ⁻⁴ M at pH 7.4, and the km value for L-aspartic acid is 2.9 × 10 ⁻² M at pH 7.4.

(3) Measurement of titer

The titer of the enzyme of the present invention was measured by an oxygen electrode method. That is, 1m1 of 0.1 M potassium phosphate buffer (pH 7.4) containing 10 mM sodium L-glutamate was placed in an oxygen electrode cell, and 10 μl of enzyme solution was added to measure the oxygen consumption rate. The amount of enzyme that consumed 1 mol of oxygen at 30 ° C. for 1 minute was set to 1 unit (Unit: abbreviated as “U” in the present specification).

In addition, since the dissolved oxygen concentration of the reaction solution decreases with the increase in temperature, the above method cannot be used for measuring the titer at a high reaction temperature. In this case, it is carried out by the MBTH method (see Anal. Biochem., 25, 228 (1968)). That is, the reaction solution containing sodium L-glutamate, catalase and the enzyme of the present invention is incubated for 20 minutes at an appropriate temperature, trichloroacetic acid (TCA) is added to stop the reaction, and the reaction stop solution is diluted with acetic acid buffer (pH 5.0). ) And 3-methyl-2-benzothiazolinonehydrazone hydrochloride (MBTH) were added, incubated at 50 ° C for 30 minutes, cooled to room temperature, and the absorbance at 316 nm was measured, and the α-ketoglu produced in the calibration curve. Quantify taric acid.

(4) optimal

The optimum pH is around pH 7-8.5 as shown in FIG. Determination of enzymatic activity at each pH was performed using sodium L-glutamate as a substrate, 0.2 M acetic acid buffer (pH 3.5-6.0), 0.2 M potassium phosphate buffer (pH 6.0-8.5) and 0.2 M glycine-sodium chloride as buffers. -Sodium hydroxide (pH 8.5-12.0) was used at 30 degreeC.

In addition, FIG. 1 is cited in FIG. 1 of the present invention enzyme (solid line) and the known enzyme (dotted line: Japanese Patent Application Laid-Open No. 57- (1982) -43685 for comparison of proper pH of the present enzyme and known enzyme). PH activity curves are written together.

As is apparent from FIG. 1, the enzyme of the present invention differs from a known enzyme even at an optimum pH.

In addition, the pH range of the case of using aspartic acid as a base is narrow and the optimum pH is 7-8. However, at pH 6.0 and above 10.0, it has little effect on L-aspartic acid (activity of glutamic acid at pH 6.0). 0.1% or less).

(5) pH stability

After maintaining at 37 ° C. for 60 minutes, 45 ° C. for 15 minutes and 60 ° C. for 15 minutes at each pH of pH 3.5-11.5, the enzyme activity against glutamic acid was measured at pH 7.4. As a result, it is stable in the range of pH 5.5-10.5 in the conditions hold | maintained at 37 degreeC for 60 minutes (solid line of FIG. 2), and is stable in the range of pH 5.5-9.5 when it is the condition of hold | maintain for 15 minutes at 45 degreeC, (FIG. 3) It is stable in the range of pH 5.5-7.5 on the conditions of the hold | maintenance for 2 minutes at 60 degreeC (FIG. 4).

In addition, in FIG. 2, the pH stability curve of the known enzyme (in Japanese Patent Application Laid-Open No. 57 (1982) -43685) is shown as a dotted line for comparison of pH stability of the enzyme and known enzyme of the present invention. ) Is combined with that of the present invention.

As apparent from FIGS. 2, 3, and 4, when comparing the stable pH ranges of the enzymes of the present invention and known enzymes, the two are distinctly different, and the former is more stable in a wider pH range than the latter. have.

(6) range of suitable working temperature

At each temperature of 30 ° C-80 ° C, the reaction was performed for 20 minutes using sodium L-glutamate as a substrate, and the enzyme activity was measured by the MBTH method.

As a result, the suitable operating temperature of the enzyme of the present invention was in the range of 30-60 ° C., and the optimum operating temperature was around 50 ° C. (FIG. 5).

(7) thermal stability

After maintaining for 15 minutes in each condition of pH 5.5, pH 7.5, and pH 9.5 at each temperature of 40 degreeC-90 degreeC, the enzyme activity with respect to glutamic acid was measured at pH7.4.

As a result, in pH 5.5, it is stabilized to 65 degreeC and shows the remaining activity of about 50% at 85 degreeC (FIG. 6, ▲-▲). In pH 7.5, it is stable up to 50 degreeC, and shows the remaining activity of about 60% at 75 degreeC (FIG. 6,-). It is stable up to 45 degreeC also in pH 9.5, and shows about 50% of residual activity at 70 degreeC (FIG. 6, ■-■).

And for comparing the thermal stability of the present invention enzyme and known enzyme, the temperature stability curve of the known enzyme (cited in FIG. 3 of Japanese Patent Application Laid-Open No. 57 (1982) -43685, indicated by a dotted line) In parallel with that of the enzyme.

As is apparent from FIG. 6, the enzyme of the present invention has higher temperature stability than the known enzyme.

(8) inhibition, activation and stabilization

In order to investigate the effects of the various additives on the enzyme of the present invention, the enzyme reaction was carried out in a reaction solution (pH 7.4) containing 1 mM of each substance shown in the second table.

The results are shown in Table 2.

[Table 2]

EDTA: Ethylenediaminetetraacetic acid

2) NEM: N-ethylmaleimide

3) PCMB: Parachloromercurybenzoate

4) DDTC: diethyldithio carbamate

5) Tiron: 4, 5-dihydroxy-1, 3-benzenedisulfonic acid disodium salt

As is clear from Table 2, the enzymes of the present invention are inhibited by about 45% by parachloromercury benzoate, but not at all by cupric chloride and diethyl dithiocarbamate. On the other hand, known enzymes are completely inhibited by dichlorodithiocarbamate chloride. Therefore, both enzymes are also different in that they are influenced by an inhibitor. In addition, activators and stabilizers of the enzymes of the present invention have not yet been found.

(9) UV absorption spectrum (see Figure 7)

λ _max : 273nm, 385nm, 465nm Shoulder: around 290nm, around 490nm

(10) coenzyme

The supernatant obtained by subjecting the enzyme of the present invention to heat treatment or trichloroacetic acid (TCA) treatment and centrifugation, the absorption was consistent with plyvin adenine dinucleotide (FAD), and activated the apo-effect of D-amino acid oxidase. It has been found that the coenzyme of the enzyme of the present invention is FAD. In addition, it is also identified as FAD from the Rf value in thin layer chromatography. FAD was estimated to exist 2 moles per mole of the enzyme of the present invention.

(11) polyacrylamide gel electrophoresis

The purified enzyme of the present invention showed a single band.

(12) molecular weight

The molecular weight of the enzyme of the present invention was determined to be 135.000 ± 10,000 in gel filtration by Sephadex G-200 (manufactured by Pharmacia Fine Chemicals).

(13) isoelectric point

As measured by isoelectric point electrophoresis using Ampoline (manufactured by LKB), pI was 6.2.

(14) Crystal structure and elemental analysis

Since the enzyme of the present invention was not crystallized, it was not measured.

(15) Resetting Method

The production method of the enzyme of the present invention can be appropriately combined with a salting method, an isoelectric point precipitation method, an organic solvent precipitation method, an adsorption method with diatomaceous earth, activated carbon, and various chromatography methods. Specific examples of the purification method are as shown in Example A1.

(B) Preparation of the enzyme of the present invention

Next, the method for producing the enzyme of the present invention by culturing the microorganism will be described in detail.

Used microorganism

The microorganisms used in the preparation of the enzymes of the present invention belong to the genus Streptomyces and are microorganisms having the ability to produce the enzymes of the present invention. Specific examples of such microorganisms are isolated from the soil of Tonosho-machi, Chibaken Katori-gun, Japan, and isolated X-119-6 strains as a single strain. The bacteriological properties of this strain are as follows.

A microscopic observation

The aerial mycelia (氣 菌 5) are linear, the width of which is 0.9-1.0 µ, and represents a simple branch. Spore disease (胞子 柄) consists of a chain of a large number of spores, forming a spiral of 2-5 rounds. The spores are slightly elliptical, the size is 0.9-1.0 × 1.1 × 1.2μ, and the electron microscope shows that the spores have a visible structure on the surface. No fragmentation of the mycelia was observed.

B. Visual Observation

The result of visual observation of the growth (30 degreeC, 16 days culture) in various media is as follows.

(1) sucrose nitrate agar medium

Its growth is poor. The mycelia are grayish brown and do not penetrate into the agar. The mycelia are powdery and spread radially thinly on the agar. The color is grayish brown and forms gray spores. No production of pigment into the medium was seen.

(2) Glucose Asparagine Agar Medium

The growth is good. The mycelia are white yellow, invading into the agar and at the same time rising slightly. The mycelia were poor in white, and no pigment was observed in the medium.

(3) Glycerin Asparagine Agar Medium

The growth is good. The mycelia are white yellow and invade into the agar and rise at the same time. No aerial mycelium was formed, and no production of pigment into the medium was observed.

(4) starch inorganic salt agar medium

The growth is good. Bacillus mycelia are white yellow and invade into the agar and rise at the same time. Bacillus mycelia are white and abundant, and gray spores grow. No pigmentation was found in the embryo.

(5) ditocin agar medium

Its growth is very good. The mycelia are white yellow. Bacillus mycelia are rich in white and greyish spores. No production of pigment into the medium was seen.

(6) Impacted Agar Badge

Its growth is very good. The mycelia are white yellow and invade into the agar and rise at the same time. The aerial mycelia are white, and no spore engraftment is seen. The production of pigments into the medium is also not seen.

(7) East Malt Agar Badges

Its growth is very good. The mycelia are white yellow and invade into the agar and rise at the same time. The aerial mycelia are white and abundant, and greyish spores are grown. No production into the medium is seen.

(8) Oatmeal Agar Badge

Its growth is very good. The mycelia are white and invade into the agar, but do not rise above the medium. Bacillus mycelia are white and abundant, greyish spores. The production of pigment into the medium is not seen.

C. Physiological Properties

The growth temperature range is 8-40 ℃, near 35 ℃ is suitable. In both the cytosine agar medium and the peptone-east-iron-agar medium, no pigments such as melanin are produced, and the gelatin is slightly liquefied and the starch is hydrolyzed.

D. Assimilation of Various Carbon Sources

The availability of the various carbon sources on the Friedham-Gottrib agar medium is shown in Table 3.

[Table 3]

* +: Used

-: Not used

Summarizing the appearance of normal is as follows. That is, the pseudohyphae is helical, and the surface of the spores is spiny. The growth on the medium showed white yellow or grayish brown, the mycelia were white to grayish brown, and no soluble color and production of pigments such as melanin were observed. In addition, starch decomposability is strong. Based on these results and the assimilation of the carbon sources shown in Table 3, according to the classification system in the 8th edition of Burgy's Manual of Determinative Bacteriology (1974) As the strains were identified, this strain belongs to the genus Streptomyces, but no known species sufficiently consistent with the characteristics of the strains were not found, and this strain was identified as a new strain and Streptomyces sp. Named X-119-6 (Streptomyces spX-119-6).

About this strain, it was entrusted to 1-3 microbial industrial technology research institute (FRI) 1-3 Yatabe-machi, Ibagaki Kentsukuba-gun, Japan on June 5, 1982, and FERM P-6560 is given as a deposit number have. In addition, the strain was delivered directly from the Japanese FRI to Rockville Parkron Drive 12301, American Type Culture Collection, United States of America, and the microorganisms were deposited and received accession number ATCC 39343 on April 26, 1983. . In addition, the Streptomyces sp X-119-6 strain was delivered directly to the Korea Advanced Institute of Science and Technology from the Japan Institute of Industrial Technology and Microbiological Technology Research Institute and deposited with the Korean Institute of Science and Technology on September 5, 1983. KAIST 830905- Deposited as 10715.

The strain is an example of a strain having high production capacity of the enzyme of the present invention, and the microorganism used in the present invention is not limited thereto. Then, the enzyme-producing bacterium of the present invention is mutated by a conventional microbial mutation induction method, for example, physical treatment such as ultraviolet ray, x-ray, r-ray irradiation, and treatment by medical chemical treatment such as nitrosoguanidine or the like. Any of the highly productive mutant strains of the obtained enzyme of the present invention can be suitably used. In addition, the object of the production method of the present invention is basically to use the function of synthesizing the protein by the genetic DNA responsible for the genetic information relating to the production of the enzyme of the present invention of the microorganism. Therefore, such genetic DNA is put into an appropriate vector and transferred into a microorganism other than the above by transformation, or the genetic DNA is introduced into another genus microorganism by cell fusion by protoplast method. The manufacturing method of the enzyme of this invention by the microorganism obtained by the integrating method is also included in the scope of the present invention.

Cultivation Methods and Conditions

The method and conditions for culturing the microorganisms used are not particularly limited as long as the microorganisms grow well and the enzymes of the present invention are sufficiently produced, but a solid culture method or a method equivalent thereto is suitable.

The solid medium used for the solid culture is not different from that usually used. That is, the solid medium mainly consists of a single solid or a combination of two or more kinds of natural solids such as bran, skim soybean, rice bran, corn, rapeseed, wheat, rice, rice husk, and if necessary, the use of the present invention. Nutrients that microorganisms can magnetize, such as glucose, sucrose, arabinose, fructose, mannitol, inositol, soluble starch, carbon sources such as ethanol, various amino acids, peptone, soy flour, Protein hydrolysates, cornspikers, gorekis, yeasts, various ammonium salts, various nitrates, nitrogen sources such as urea, various sodium salts, potassium salts, calcium salts, manganese salts, magnesium salts, zinc salts, iron salts, phosphate sulfates the no-no acid, cyano Noko soften non such as growth medium with appropriately adding a cow, or an appropriate combination thereof, size, medium, etc. made of the particles of the ^form-such as salts, diamine, riboflavin, niacin, pantothenic acid, biotin, p . Such a solid medium is sterilized or denatured by a conventional method, and the seed is inoculated to carry out solid culture.

In addition, even if the culture method other than the above, for example, by absorbing or coating the liquid medium in a suitable carrier such as a sponge (see Japanese Patent Application Laid-Open No. 49 (1974) -14679), inoculation and incubation of the seed microorganisms are used. As long as it breeds and produces the enzyme of this invention favorable, it can employ | adopt. The culture conditions may be selected in accordance with the type of microorganism used to best suit the conditions for the production of enzymes, and is not particularly limited. Usually, what is necessary is just to culture | cultivate for 5-15 days, for example at 20-30 degreeC and pH 5-7.

Sampling of the Enzyme of the Invention

The enzymes of the present invention produced by the culturing of the microorganisms used are extracted and separated from the culture, i.e., the medium and / or the culture by a suitable extraction method. It is used as a crude enzyme solution as it is, or it is refine | purified by the refinement | purification stage suitable for a use purpose according to a conventional enzyme purification method as mentioned above. The extraction method is not particularly limited and is performed by a conventional method. For example, extraction from solid cultures is usually done with water or buffers. In addition, the enzyme of the present invention in cells is crushed, solubilized and extracted by a conventional method.

(II) Analysis of L-Glutamic Acid

1) Analytical Sample

In the present invention, the term "analytical sample" (A) is a sample containing or expected to contain L-glutamic acid as its component, and a sample to analyze the content of L-glutamic acid or the presence or absence thereof, or (B) A sample containing a reaction system which is expected to liberate or liberate L-glutamic acid, and by measuring a change in the L-glutamic acid content of the system, the enzyme activity involved in the system or L- in the system It means a sample for analyzing the content of substances to be converted into glutamic acid or the presence or absence of these substances.

Sample sauces classified in the above (A) are foods (e.g., soy sauce, amino acid seasonings, various seasonings, liquid seasonings such as liquid soup flavors, alcohol-containing foods such as sake, sake, sake products, sausages, Biological samples (eg, urine, blood, etc.) such as extracts of solid foods such as ham, and the like, and the samples classified into (B) include glutaminase, glutamic acid racemase, GOT, GPT, and r-GTP. -A system containing an enzyme which uses glutamic acid as a reaction product and its substrate, or a system containing one or two or more different conjugated enzyme systems different from the above-mentioned systems.

2) Analysis of L-Glutamic Acid

In the present invention, the analysis method of L-glutamic acid is pH5-9 in the enzyme reaction system of the enzyme of the present invention of L-glutamic acid in the sample, particularly pH 5-6 for L-glutamic acid in the sample in which a large amount of L-aspartic acid coexists. The enzyme reaction system in the present invention and the detection system for the target substance which performs quantitative or normal detection of the target substance consumed or produced accordingly.

Enzyme reaction

The enzyme reaction conditions in the analysis of L-glutamic acid are as follows. In other words, the reaction pH should be a pH that does not deactivate the enzyme of the present invention and fully functions against L-glutamic acid, and if the detection system of the target substance depends on pH, the pH is set in consideration of such conditions. It is desirable to. Usually, enzyme reaction is performed in pH5-9. In the case where the sample contains a large amount of L-aspartic acid that substantially affects the analysis of L-glutamic acid according to the present invention, the enzyme of the present invention acts somewhat against L-aspartic acid at an appropriate pH, so that L- By reacting with glutamic acid to a degree sufficient for detection of a target substance and reacting with conditions that do not act with L-aspartic acid, even such a sample can specifically analyze L-glutamic acid. Under these conditions, a reaction condition of pH 5-6 is appropriate.

In addition, for the purpose of maintaining the pH of the enzyme reaction in an appropriate range, it is preferable to use various buffers as the reaction medium. As the buffer solution, it is possible to maintain the pH range, and not to inhibit the enzyme of the present invention, and not to affect the detection system of the target substance. For example, phosphate buffer, tris hydrochloric acid buffer, acetic acid buffer, citric acid buffer, bellanal buffer and the like are exemplified.

The reaction temperature is not particularly limited. In view of the optimum temperature of the enzyme of the present invention, the stable temperature, the detection system of the target substance to be used and the like, any person skilled in the art can easily determine the result. In the reaction, the enzyme of the present invention is used as an immobilized enzyme immobilized by a general method such as a soluble enzyme or a comprehensive method, an adsorption method, a crosslinking method, and a covalent bonding method.

The immobilization state is not particularly limited (see "Immobilization Enzyme" Chihiro Ichita, published by Godansha, March 20, 1975). For example, the material of the immobilization carrier is cellulose (cellophane, filter paper), cellulose derivatives including acetylcellulose derivatives, nitrocellulose (collodione), various ion exchange cellulose derivatives (DEAE-cellulose, TEAE-cellulose, ECTEOLA-). Cellulose, CM-cellulose, P-cellulose), starch, dextrin derivatives (Sephadex), agarose, mannan (conjacmannan), chitosan, carrageenan, alginic acid, xanthan gum, polysaccharides such as agar , Gelatine, fibroin, keratin, albumin, guten and other proteins, polyacrylamide, polyvinylpyrrolidone, polyvinyl alcohol and the like, polymer gels, polyvinyl chloride, polymethylmethacrylate, polyacrylonitrile, polystyrene, poly Aminostyrene, polyethylene, polypropylene, polyurethane, polycarbonate, polyamide (nylon, polyamino acid), fluororesin (Teflon), silicone water Synthetic organic polymers such as resins, photosensitive resins, adsorptive resins, ion exchange resins, inorganic polymers such as glass, silica, ceramics, alumina, kallinite, bentonite, potassium phosphate, hydroxyapatite, activated carbon, etc. May be used as is or after introduction of a functional group capable of reacting with an enzyme protein or activation of the functional group.

The enzyme may be polymerized, gelled, cellulose derivatives, polysaccharides or proteins, microcapsules, organic polymers or cellulose derivatives, etc. And immobilized by covalent bonding to a carrier by an alkylation method or a crosslinking method (by glutaraldehyde, hexamethylene diisocyanate, etc.), ionic bonding to a carrier having an ion exchange group, or physical adsorption. Further, according to the crosslinking method, glutaraldehyde, isocyanate derivatives (hexamethylene diisocyanate, toluene diisocyanate and the like), isothiocyanate derivatives (hexamethylene diisothiocyanate and the like), N, N'-ethylene Bismaleimide, N, N '-(1,2-phenylene) bismaleimide, N, N'-(1,4-phenylene) bismaleimide and N, N'-poly It can be immobilized by crosslinking between enzymes using a reagent having two or more functional groups such as methylene-bisiodoacetamide. Immobilized enzymes can also be prepared by combining two or more of the above methods.

According to the above method, the enzyme of the present invention can be immobilized, and can be prepared in a suitable form such as thin film, gel, granule, powder, microcapsules, tube, fiber, hollow fiber and container.

Detection system of target substance

The target substances for analyzing L-glutamic acid by the method of the present invention are oxygen consumed by the enzymatic reaction, hydrogen peroxide produced, ammonia and α-ketoglutaric acid. Detection of each target substance is carried out by an arbitrary method, and the present invention is not limited to the detection method. That is, although a well-known method is employ | adopted as a detection method normally, it is thought that various methods further developed in the future can also be employ | adopted. Next, the detection method of each target substance is demonstrated.

(1) detection of oxygen

As a method of detecting the consumed oxygen, the Würburg test method (Biochemistry Experiment Lecture 5, Enzyme Research Method, pages 35-41, Tokyo Kagakudojin Co., Ltd., issued August 20, 1975) and the oxygen electrode method (by electrode method) Coral measurement, Hagiwara basin piece, Godansha Co., Ltd., issued November 20, 1977) are known, and a freezing method may be employed in the present invention.

As the electrode used in the oxygen electrode method, platinum, gold, iridium and the like are used as working electrodes, and electrodes such as silver, silver / silver chloride, saturated caltomel, lead, zinc, and aluminum are used as manufacturing electrodes. The test liquid of an electrode and electrolyte solution (alkali solution, such as potassium hydroxide and sodium hydroxide) is taken as a basic structure. Complex electrodes (Fig. 17) or separate electrodes, such as a Clark type electrode, are used. The method may be a polar graph method or a galvanic cell method. In the measurement of oxygen by an oxygen electrode, the enzyme half may be measured by the conventional method of dissolved oxygen of the mixture. In addition, you may measure using an enzyme electrode.

(2) detection of hydrogen peroxide

As a method of detecting the generated hydrogen peroxide, electrochemical analysis, spectroscopic analysis, fluorescence analysis, chemiluminescence analysis and the like are known, and any method may be employed in the present invention.

As an electrochemical analysis method, it is common to measure by an electric current method using the hydrogen peroxide detection electrode of the same structure as the said oxygen electrode. For example, the method of using the working electrode which consists of a noble metal electrode, such as platinum, a control electrode which consists of silver electrodes, etc., and the Clark type hydrogen peroxide electrode which consists of a hydrogen peroxide permeable membrane is used suitably for this invention. Moreover, you may use as an enzyme electrode like an oxygen electrode. In addition to the above-described methods, hydrogen peroxide and iodine ions are reacted with an enzyme of the present invention using a catalyst for decomposition of hydrogen peroxide, such as peroxidase or molybdate, together with the enzyme of the present invention to reduce the amount of iodine ion active on the surface of the electrode. The measuring method can also be used. In addition, this method is suitably used as an enzyme electrode equipped with an iodine ion electrode and an immobilized enzyme containing the enzyme, peroxidase or catalyst of the present invention.

Spectroscopic methods include (1) a peroxidase method in which an oxidative chromophore is reacted with hydrogen peroxide in the presence of a peroxidase or a substance exhibiting the same activity to measure the absorbance of the resulting color, and (2) hydrogen peroxide in the presence of catalase. A catalase method which induces an aldehyde produced by reacting with a chromophore and measures the absorbance of the produced color, or measures the production of reduced NAD (NADH) by reacting an aldehyde dehydrogenase in the presence of NAD, and (3) There are chemical methods using Ti (IV) / xylenol orange series, Ti (IV) / 4- (2-pyridylazo) resorcinol series, V- (V) / xylenol orange series and the like.

In the peroxidase method of (1), the color reaction is an oxidation reaction of a coloring agent alone or an oxidation condensation reaction with a coupler of the coloring agent. As the former method, as a coloring agent, 0- dianisidine, 0-tolidine, 0-toluidine, 0-aninophenol, 2, 4-dichloroindophenol, benzidine, 3, 3 ', 5, 5'-terraalkyl Benzidine (3, 3 ', 5, 5'- tetrabenzidine, etc.), 4-methoxy-1-naphthol, 2,2'-azino-di (3-ethylbenzothiazoline-6-sulfonic acid), guiacum Resin (Guiaacol), N- (4-antipyryl) -aniline derivative (N- (4'-antipyryl) -2-carboxy-4-hydroxyaniline etc.), p-hydroxyphenyl acetic acid, N , N-dimethyl-p-phenylene diamine and the like can be used.

Moreover, as a latter method, 4-amino antipyrine derivatives, such as 4-amino antipyrine and 4-amino antipyrine amide; Phenylenediamine derivatives such as 4-aminophenazone, 4-amino-N, N-dimethyl aniline, p-phenylenediamine, 4-amino-N, and N-diethyl-m-toluidine; 3-methyl-2-benzothiazolinonehydrazone (MBTH) and the like are used, and as a coupler, phenol, catechol, resorcin, hydroquinone, cresol, guaiacol, pyrogallol, orcinol, p-chlorophenol, p Bromophenol, 2, 4-diclotophenol, 2, 4-dibromophenol, 2, 4, 6-tribromophenol, aniline, N, N-dimethylaniline, N, N-diethylaniline, N-ethyl (N-3-methylphenyl) -N-acetylenediamine, 3-acetamino-N, N-diethylaniline, N, N-diethyl-m-toluidine, N-ethylN- (2-hydroxy -3-sulfopropyl-m-toluidine, N-glysyl-N-ethyl-m-toluidine, p-dimethylaminophenol 0-aminophenol, m-aminophenol, p-methylaminophenol, 2-chloro-6- Phenol-based, aniline-based or toluidine-based compounds such as methylphenol, 4-chloro-3-methylphenol, 3,5-difluoro-2-hydroxybenzenesulfonic acid, and 4-halogeno-1-naphthol-2-sulfonic acid (4-chloro-1-naphthol-2-sulfonic acid, etc.), 1-naphthol-2-sulfonic acid, 2-amino-5-naphthol-7- Naphthol-based compounds such as phonic acid, 2,4-dichloro-1-naphthol, 1-hydroxy-2-naphthoic acid, 4,5-dihydroxy-2, 7-disulfonic acid, naphthylamine or derivatives thereof; Quinoline compounds, such as oxyquinoline and aminoquinoline, can be used.

Suitable combinations of colorants and couplers include combinations of 4-aminoantipyrin with phenol, N, N-dimethylaniline, N, N-diethymaniline or N-diethyl-m-toluidine or 3-methyl-2-benzothiazoli And combinations of nonhydrazone with N and N-dimethylaniline. The present invention is not limited to these types of colorimeters or colorants and couplers, and any one can be used as long as it can be quantitatively oxidized to exhibit color change. In addition, although peroxidase is a horseradish or sweet potato-derived enzyme, a peroxidase-like activity is usually used, and the peroxidase is not particularly limited. And a catalyst exhibiting the same activity as peroxidase.

In the catalase method of (2), methanol is usually used as an alcohol, and formaldehyde produced by reaction with hydrogen peroxide is oxidized (e.g., sodium periodate, potassium cyanophenolate, Hydrazone (eg 3-methyl-2-benzodiazolinonehydrazone, 4-amino-3-hydrazino-5-mercapto-1, 2, 4-triazole in the presence of ferric chloride, etc.) (AHMT, etc.), and the method of coloring, and the method of coloring by reacting formaldehyde with acetylacetone and ammonium.

Then, a method of inducing hydrogen peroxide into oxidized glutadione by glutathione peroxidase, reducing this by glutadione reductase in the presence of NADPH, and measuring the oxidation amount of NADPH (Anal. Biochem, 76, 184). -191 (1976)), and isoion-histamine-based oxidative bleaching, and there is also a method for quantifying the loss of chromaticity.

In fluorescence analysis, homovanic acid is reacted with hydrogen peroxide in the presence of peroxidase to convert to fluorescent 2, 2'-dihydroxy-3, 3'-dimethoxybiphenyl-5,5'-diacetic acid, and the fluorescence intensity In the same manner, p-hydroxyphenyl acetic acid, diacetyl fluorescin derivatives (diacetyl fluorescin, diacetyl dichloro fluorescin, etc.) are emitted instead of homo vanillic acid. You may use as a reagent. There is also a method of oxidizing fluorescent substances such as scopoline, 3, 5-diacetyl-1, and 2-dihydrolutidine by oxidizing with hydrogen peroxide / peroxidase, converting them into non-fluorescent substances, and measuring the decrease in fluorescence. .

As an optical luminescence analysis method, there is a method of reacting luminol with hydrogen peroxide in the presence of peroxidase and measuring the amount of emitted light (see Japanese Patent Laid-Open No. 57 (1982) -71399 and Japanese Patent Laid-Open No. 57 (1982) -71400). In the same way, isoluminol, pyrogallol, bis (2, 4, 6-trichlorophenyl) oxalate may be used instead of luminol, and hemoglobin, hematin, hemin, iron (c) potassium cyanide, cobalt chloride instead of peroxidase. Etc. may be used as a catalyst.

(3) detection of ammonia

Ammonia can be analyzed by methods such as the micro-kjeldahl method, the Nessler method, the indophenol method, the ninhydrin method, the phenosafranin method, and the like. In addition, ammonium ions can be analyzed by a cation selective electrode. In addition, the analysis may be performed by an electrochemical analysis method, such as a method for analyzing ammonium ions by a cation-selective electrode or a method for analyzing ammonia gas by an ammonia gas electrode composed of a glass electrode equipped with a hydrophobic gas permeable membrane. do. And amnonia may be detected by the enzyme electrode which combined these electrodes and the enzyme of this invention, and L-glutamic acid may be analyzed.

(4) Detection of α-ketoglutaric acid

α-ketoglutaric acid is reacted with 3-methyl-2-benzothiazolinone hydrazone (MBTH) to measure the absorbance of the product, and 2,4-dinitrophenylhydrazine is reacted to measure the absorbance of the product , By measuring the absorbance of the product by reacting with 0-phenyllandiamine, and other known methods can be used.

3) Reagent for analysis

The analytical reagent of the present invention is a reagent containing at least the enzyme of the present invention. That is, the form is not limited, and may be a soluble enzyme of a solution, powder or granules, and may be applied to a carrier of a thin film, gel, granule, powder, microcapsules, tube, fiber, hollow fiber or container by various methods as described above. The immobilized immobilization enzyme may be sufficient. In addition to the enzymes of the present invention, in addition to liquid or phosphate buffers, tris hydrochloride buffers, acetic acid buffers, citric acid buffers, chloronal buffers, buffers, salts (such as sodium chloride), sugars (such as sucrose), polyhydric alcohols (glycerol, propylene glycol, sorbitol, etc.). ), Other suitable stabilizers, surfactants and the like may be added.

In the analysis of L-glutamic acid, the analytical reagent is used so that the required enzymatic activity is obtained according to the various detection methods. In addition, you may enclose in advance the quantity suitable for each detection method in containers, such as a reagent bottle and an ampule.

4) Analysis Kit

The kit for analysis consists of an assay reagent containing the enzyme of the present invention and a reagent for detection which is a means for detecting a reaction by the enzyme of the present invention. The detection reagent is a reagent necessary for detecting the target substance. In other words, when hydrogen peroxide is used as a target, detection reagents, for example, an active agent such as a peroxidase or a peroxidase, a combination of a colorant or a colorant and a coupler, an active agent such as a catalase or a catalase, and an alcohol and a color system are necessary. Combinations of reagents required for reagents or conjugated enzyme systems, combinations of active agents such as peroxidases or peroxidases with luminescent agents, combinations of active agents such as peroxidases or peroxidases with luminescent reagents, and the like. Specific examples of these reagents are apparent from the description of "detection of hydrogen peroxide" above.

Similarly, in the case where ammonia or α-ketoglutaric acid is used as the target substance, the necessary detection reagent may be combined with an analytical reagent containing the enzyme of the present invention and configured as an analytical kit. The reagent for analysis containing the enzyme of the present invention and the reagent for detection may be mixed with each other to form a single reagent, and when there are components that interfere with each other, the components may be divided so as to be in an appropriate combination. And these may be prepared as a solution form or a powdery reagent, and may be contained in a suitable support, such as a filter paper or a film, and may be prepared as a test paper or an analytical film. In addition to the combination reagent, a standard reagent containing a predetermined amount of L-glutamic acid may be added to the analysis kit of the present invention.

One suitable example of the analytical kit of the present invention is a kit for analyzing L-glutamic acid by spectroscopic detection of hydrogen peroxide. For example, in the case of a kit by a peroxidase method, at least 0.02 unit (U) of enzyme of the present invention / test, peroxidase 1-10U / test, 4-amino antipyrine and phenol or N, N-dimethylaniline as a colorant When used, these reagents are used at least 1 mole, preferably at least 2 moles, relative to the hydrogen peroxide produced.

(Ⅲ) biosensor

1) Configuration of Sensor

In the present invention, a "biosensor" is a transducer that is a signal conversion site for detecting an enzyme and a chemical or physical change in an analyte sample by the action of the enzyme, and converting an electrical signal. Means a device for measuring an electrical signal as a change in electric current or a change in potential, and analyzing the amount or presence of a detected substance in an analytical sample ("Chemical Area, Intermediate 134, Biomaterial Science Vol. 1, pp. 69-79, issued April 20, 1982, published by Nankodo Co., Ltd. and the Chemicals, June 1982, pages 491-496. As described above, a biosensor using an enzyme as a receptor portion is also called an "enzyme sensor", and in particular, an enzyme sensor using an electrochemical device as a transducer portion is called an "enzyme electrode" ("ion electrode and enzyme electrode", Shuichi Suzuki, pp. 65-106, published November 1, 1981, issued by Godansha and "The Field of Chemistry", Vol. 36, No. 5, pages 343-349 (1982). In addition, the enzyme sensor includes an electrode-mounted enzyme electrode configured to be in close or close proximity to the receptor portion and the transducer portion, and a reactor-type enzyme sensor configured to separate the receptor portion and the transducer portion.

The biosensor according to the present invention constitutes a novel biosensor by using a specific L-glutamic acid oxidase, which is a novel enzyme, as a receptor in the known biosensor, and achieves a distinctive effect in comparison with the prior art. Therefore, in this invention, a well-known technique can be employ | adopted except for using the enzyme of this invention, and the technique to be developed in the future can also be employ | adopted if it meets the objective of this invention.

2) Receptor part

In order to use the enzyme of the present invention as the receptor portion of the biosensor, the contact between the enzyme of the present invention and L-glutamic acid, which is a detection substance, is easily performed, and the enzyme reaction proceeds smoothly, and the enzyme of the present invention is analyzed. The receptor portion must be configured in a state that does not substantially elute during the process. Therefore, the present invention is usually used by being fixed or coated with a semipermeable membrane (for example, dialysis membrane, ultrafiltration membrane, etc.).

Immobilization may be made in a state suitable for the purpose of use in the biosensor of the receptor portion, and the enzyme of the present invention may be inactivated substantially and the enzyme reaction may not be inhibited. "Fixing Enzyme", published by Godansha Co., Ltd., March 20, 1975, and "Ion Electrode and Enzyme Electrode" (simultaneous publication), pages 65-77).

That is, the immobilization method is immobilization used as a receptor portion by a known method such as a comprehensive method (lattice method, microcapsule type, etc.), carrier bonding method (covalent bond method, ion bonding method, physical adsorption method, etc.), crosslinking method, and the like. What is necessary is just to select the suitable method as an enzyme preparation method. Specifically, materials suitable for the material of the carrier or the support are as described above.

The form which uses the obtained immobilized enzyme as a receptor part of a biosensor is as follows. That is, in the case of an electrode-mounted enzyme electrode, a membrane, gel, powder, or microcapsule-immobilized enzyme is usually attached to or close to the working surface of the measuring electrode, and preferably, a substrate-permeable porous membrane (e.g., dialysis membrane, ultrafiltration membrane). ) Is coated. The three members of the electrode membrane, the immobilized enzyme layer, and the coating membrane in contact with the sample liquid may be composed of an integral network or heterogeneous membrane in which all are integral, or a laminated membrane or heterogeneous membrane in which the former two or the latter two characters are integrated. good. In the case of a reactor-type enzyme sensor, a granular or powdery immobilized enzyme is usually filled with a column or a tube or fibrous (hollow-fiber) immobilized enzyme can be configured as a reactor and used as a receptor portion.

3) Transducer

In the receptor section, the transducer section is a site for oxidizing the substrate in the analytical sample by the enzyme of the present invention, detecting chemical or physical changes caused by the reaction, and converting the substrate into an electrical signal. Although the case where a chemical change is detected in this invention is illustrated, physical change, for example, heat resin may be detected by a thermistor etc.

The chemical change accompanying the reaction by the enzyme of the present invention is a decrease in oxygen and L-glutamic acid and an increase in hydrogen peroxide, ammonia and α-ketoglutaric acid in the analytical sample, but the absorption of oxygen usually detected by the transducer part , Increase in hydrogen peroxide or increase in ammonia. An oxygen electrode is used as a transducer which detects the absorption of oxygen and converts it into an electrical signal. As a transducer for detecting hydrogen peroxide and converting it into an electrical signal, a hydrogen peroxide detection electrode is used. Specifically, an electrode having the same structure as the oxygen electrode may be used. In addition, hydrogen peroxide can be detected by a method of detecting the reduction in the amount of hydrogen peroxide and iodine ion using a catalyst for decomposition of hydrogen peroxide, such as peroxidase or molybdate, together with the enzyme of the present invention as the receptor portion. In this case, an iodine ion electrode can be used.

As a transducer that detects ammonia and converts it into an electrical signal, a cation selective electrode can be used when analyzing ammonia as ammonium ion, and a glass electrode equipped with a hydrophobic hydropermeable membrane when analyzing ammonia gas. An ammonia gas electrode can be used. The electrode-mounted enzyme electrode according to the present invention is constructed by being mounted on the electrode-sensitive material permeable membrane of the various electrodes exemplified above by the enzyme of the present invention as the receptor portion or integrally mounted with such a membrane. The reactor-type enzyme electrode according to the present invention is composed of a reactor in which the enzyme of the present invention as the receptor portion is immobilized, and various electrodes exemplified above separated from the enzyme reaction portion. In addition, the present invention is not limited to the transducer detection material and the transducer method.

Example A1

Into a 500 ml Erlenmeyer flask, 20 g of bran and 16 ml of water were placed, and Streptomyces sp X-119-6 (KAIST 830905-10715, FERM P-) was prepared in a bran medium prepared by heat sterilization at 120 ° C. for 30 minutes. 6560, ATCC 39343) were inoculated, and cultured at 28 ° C. for 7 days to prepare a seed. Each of 25 Erlenmeyer flasks for 51 was put 200 g of push and 160 ml of water, sterilized under pressure at 120 ° C. for 30 minutes, inoculated aseptically, incubated at 28 ° C. for 2 days, and then the temperature was 20 ° C. Down to 2 weeks of incubation.

The obtained culture was soaked in 37.5 L of water for 1 hour, filtered, and passed through diatomaceous earth to obtain about 34 L of crude enzyme solution. Ammonium sulfate was added to this crude enzyme solution to 50% saturation, the resulting precipitate was collected by centrifugation, dissolved in 3.9 L of 0.02M acetic acid buffer (pH 5.5), and heated to 57 ° C for 30 minutes. After the heat treatment of the enzyme solution at 5 DEG C or lower, the mixture was cooled. Two times the amount of pre-cooled ethanol was added, the resulting precipitate was collected by centrifugation, dissolved in 0.02 M phosphate buffer (pH 7.4), and dialyzed overnight with the same buffer.

The precipitate produced during dialysis was centrifuged, the supernatant was passed through DEAE (diethylaminoethyl) -cellulose column (3.5 x 50 cm) equilibrated with the same buffer, and the adsorbed enzyme was passed through the same buffer containing 0.35 M sodium chloride. Eluted using. The eluted fractions were collected and dialyzed overnight with 0.05 M acetic acid buffer (pH 5.5) containing 0.05 M of salt. The dialysate was passed through DEAE-Sepharose CL-6B (manufactured by Pharmacia Fine Chemicals) Colum (2 × 10 cm) equilibrated with the same buffer solution, and the adsorbed enzyme was passed through a linear gradient of 0.05-0.75 M of salt. The eluted active fractions were collected and concentrated by dialysis, followed by gel filtration using Sephadex G-200 (manufactured by Pharmacia Fine Chemicals) Colum (2.5 × 120 cm), and the active fractions were collected and concentrated. Dialysis was performed with 0.02 M potassium phosphate buffer (pH 7.4). The dialysis solution was centrifuged, the supernatant was subjected to microfiltration, and then lyophilized to obtain 30 mg of purified product of L-glutamic acid oxidase (inert 55.1 U / mg protein, yield 18.4%).

Example B1

(1) preparation of kits

Reagent A: 0.2 M phosphoric acid so that 1 mg (10 U / mg) of L-glutamic acid oxidase, 5 mg of peroxidase (100 U / mg: derived from Western mustard: manufactured by Toyoboseki Co., Ltd.) and 40 mg of 4-aminoantipyrine in one reagent bottle It was dissolved in 2 ml of potassium buffer (pH 6.5) and lyophilized by a conventional method. Reagent B: (1) 40 mg of phenol was dissolved in 0.1 M phosphate buffer (pH 6.5), and 100 ml was added to the reagent bottle, or (2) 100 mg of dimethylaniline was dissolved in 0.1 M potassium phosphate buffer (pH 6.5). 100 ml was added to the reagent bottle.

(2) How to operate

0.1 ml of the standard solution of sodium L-glutamate and water were each divided into 0.1 ml of the test tube, and the lyophilisate of the reagent A was dissolved in 100 ml of the solution of one of the reagents (1) or (2). It was added and incubated for 20 minutes, shaking aerobicly at 37 degreeC. In contrast to the blind test in water, the absorbance of 500 nm was measured when using (1), and the absorbance of 565 nm was measured when using (2). These calibration curves are shown in FIGS. 8 (for reagent B (1)) and 9 (for reagent B (2)).

Example B2

(1) Preparation of reagent

Color Reagent: 20 mg of phenol, 20 mg of 4-aminoantipyrine and 3 mg of Western Mustard Peroxidase (100 U / mg) were dissolved in 50 ml of 0.2 M potassium phosphate buffer (pH 7.4). L-glutamic acid oxidase: 300 g (55 U / ml) of purified enzyme was dissolved in 30 ml of 0.02 M potassium phosphate buffer (pH 7.4) (0.05 U / ml).

(2) How to operate

0.8 ml of color reagent was divided into test tubes, and 0.1 ml of L-glutamic acid oxidase solution was placed in a 37 ° C. thermostat and warmed for 5 minutes. 0.1 ml of standard L-glutamate solution (0-2 μmol / ml) or a sample solution (a solution of 150 times diluted in soy sauce of various products) was added thereto, mixed well, and incubated for 20 minutes with aerobic shaking at 37 ° C. Using the blind test as a control, the absorbance at 500 nm was measured, and a calibration curve shown in FIG. 10 was prepared. Quantitative value of L-glutamic acid in the sample obtained from this calibration curve by the conventional method using L-glutamic acid decarboxylase (method of measuring the sensitivity of phenolphthalein by Technicon automatic analyzer). It showed in the 4th table (it shows after Example B3) compared with the process. Correlation coefficient r = 0.997 and rare equation y = 1.005x-0.575 were found.

Example B3

0.7 ml of 0.1 M potassium phosphate buffer solution (pH7.4), 0.1 ml solution of chitillase (1000 U / ml from bovine liver, manufactured by Sigma Chemical) and 0.1 ml solution of L-glutamic acid oxidase (0.6 U / ml) Was taken and warmed at 37 ° C. for 5 minutes. The reaction was initiated by adding 0.1 ml of standard L-flutamate solution (0-5 mM) or 0.1 ml of sample solution (same soy diluent as in Example B2). After incubation with aerobic shaking at 37 ° C. for 220 minutes, 0.1 ml of 25% trichloroacetic acid was added to stop the reaction. 1.9 ml of 1 M acetic acid buffer solution (pH5.0) and 0.8 ml of 0.1% 3-methyl-2-benzothiazolinonehydrazone hydrochloric acid were added and mixed, followed by incubation at 50 ° C for 30 minutes. After cooling to room temperature, the absorbance at 316 nm was measured using the blind test as a control, and a calibration curve shown in FIG. 11 was prepared. The quantitative value of L-glutamic acid in the sample solution obtained from this calibration curve was compared with the quantitative value of L-glutamic acid in the sample solution by the conventional method using L-glutamic acid decarbonase, and is shown in Table 4 below. The correlation coefficient r = 0.996 and the regression equation y = 1.026x-3.122.

[Table 4]

Example B4

(1) Preparation of reagent

Color reagent: 30 mg of phenol, 30 mg of 4-aminoantipyrine, 4 mg of Western mustard peroxidase (100 U / mg) and 1 mg of L-glutamic acid oxidase (10 U / mg) were dissolved in 50 ml of 0.2 M potassium phosphate buffer (pH 6.5). Substrate solution A: 200 mg of sodium L-aspartate and 15 mg of α-ketoglutaric acid were dissolved in 10 ml of 0.1 M potassium phosphate buffer (pH 7.0). Substrate solution B: 140 mg of L-alanine and 15 mg of α-ketoglutaric acid were dissolved in 10 ml of 0.1 M potassium phosphate buffer (pH 7.0).

(2) Operation method

1) Measurement of glutamic acid-oxaloacetic acid transaminase (GOT) activity

0.2 ml of Substrate Solution A was taken into a test tube, 0.1 ml of a standard enzyme solution (380 U / mg, manufactured by Beringo Manouchi Co.) was added thereto, and after incubation at 37 ° C. for 30 minutes, 0.1 ml of 25% trichloroacetic acid was added to stop the reaction. . 0.1 ml of 1 M potassium phosphate buffer (pH6.5) and 0.5 ml of L-glutamic acid oxidase were added to the reaction stopper, and the resultant was incubated at 37 ° C for 20 minutes. Using the blind test as a control, the absorbance of 500 nm was measured and the calibration curve shown in FIG. 12 was created.

2) Synthesis of glutamic acid-pyruvic acid transaminase (GPT)

0.2 ml of Substrate Solution B was added to the test tube, 0.1 ml of standard enzyme solution (140 U / mg, manufactured by Beringa Manouchi Co.) was added thereto, and after incubation at 37 ° C. for 30 minutes, 0.1 ml of 25% trichloroacetic acid was added to stop the reaction. I was. The L-glutamic acid content in the reaction stopper was measured as in 1), and a calibration curve shown in FIG. 13 was prepared.

Example B5

1 ml of 0.1 M acetic acid buffer (pH 5.5) containing L-glutamic acid oxidase (0.5 U / ml) was sealed in a cuvette of a Clark type oxygen electrode with 30 ° C. constant temperature water circulated, and 50 ml of L-glutamic acid standard solution (0-2 mM) was injected to measure oxygen consumption, and a calibration curve shown in FIG. 14 was prepared. The L-glutamic acid quantitative value in the sample (various soy sauce) calculated | required by this calibration curve was compared with the quantitative value of L-glutamic acid in the sample by the conventional method using L-glutamic acid decarboxylase, and it is shown in the 5th table | surface. The correlation coefficient r = 0.953 and the regression equation y = 0.930x + 6.11.

[Table 5]

Example B6

0.5 ml of L-glutamic acid oxidase (12.5 U / ml) solution is dropped onto a porous nitrocellulose membrane (manufactured by Toyogaku Co., Ltd., TM-5: pore diameter 0.1 μm, diameter 25 mm, film thickness 140 μm) and absorbed by suction. It fixed and the L- glutamic-acid oxidase membrane preparation was obtained. After the immobilized enzyme membrane was cut in a circle (diameter 5.0 mm), it was mounted on a gas-permeable membrane (Teflon membrane, membrane thickness of 10 μm) of a diaphragm oxygen electrode (type U-2 manufactured by Ishikawa Seisakusho Co., Ltd.), and again the cellulose dialysis membrane was immobilized. Was mounted to produce an L-glutamic acid sensor. Using this sensor, the current reduction value for the sodium L-glutamate standard solution at a known concentration (0.05-1.0 mM) was measured, and a calibration curve shown in FIG. 15 was prepared.

Example B7

1 ml of 0.1 M acetic acid buffer (pH 5.5) containing 2 U of L-glutamic acid oxidase and 20 μmol of L-glutamic acid was sealed in a cuvette of a Clark type oxygen electrode circulated at 30 ° C., and a glutaminase standard solution (4-20 mU, 20 μl of Escherichia coli was injected to measure oxygen consumption, and a calibration curve shown in FIG. 16 was prepared. Similarly, 50 μl of suspension of soy sauce (10 g of soy sauce was homogenized in 50 ml of 0.1 M phosphate buffer (pH 7.0) at 1000 r.pm for 5 minutes and then buffered to obtain 300 ml) was injected to measure oxygen consumption. . Glutaminase activity per gram of soy sauce was measured at 3.7 U.

Example C1

0.1 ml of L-glutamic acid oxidase (448 U / ml) solution was added dropwise to a 30 mm × 30 mm square cellophane membrane (film thickness of 30 μm) and dried, and 0.1 ml of 25% glutaraldehyde solution was infiltrated thereto. It was dried overnight at 占 폚 and washed with water until the absorbent material at 280 mm was not eluted to obtain an L-glutamic acid oxidase immobilized cellophane membrane. The immobilized enzyme membrane was cut into circular small pieces and mounted on an oxygen permeable membrane (teflon membrane, membrane thickness of 10 μm) of a diaphragm oxygen electrode (manufactured by Ishikawa Seisakusho Co., Ltd.), and then on the immobilized enzyme membrane, a cellulose dialysis membrane (film thickness of 50 μm, Visking Co., Ltd.) was installed to produce an L-glutamic acid sensor (FIG. 17).

In FIG. 17, the diaphragm oxygen electrode has a platinum electrode (1) as a cathode, a lead electrode (2) as an anode, and an oxygen permeability on the surface of the platinum cathode with a potassium hydroxide solution as an internal liquid (electrolyte) 3. The teflon membrane 4 is mounted. The immobilized enzyme membrane 5 is mounted on the Teflon membrane, and is then covered with the cellulose dialysis membrane 6 and fixed with a zero ring 7.

Using the above L-glutamic acid sensor, the L-glutamic acid analyzer shown in FIG. 18 was prepared. In FIG. 18, the sample solution 2 injected from the sample solution inlet 1 is fed to the cell 5 by means of the ferritic pump 4 together with the dissolved oxygen saturation buffer 3. At this time, the current reduction value measured by the L-glutamic acid sensor 6 is recorded in the recorder 7. The L- glutamate sodium sulfate solution was continuously injected into the apparatus, the current reduction value was measured, the correlation with sodium L-glutamate concentration was determined, and the results are shown in FIG.

The responsiveness at each temperature was measured using this apparatus, and the result was shown in FIG. However, the measurement was performed at each temperature of 20 degreeC, 30 degreeC, and 40 degreeC using the 0.8 mM L- glutamate sodium solution. Similarly, the responsiveness (30 ° C.) to L-sodium glutamate solution (0.8 mM) at each pH was measured, and the results are shown in FIG. The concentration of each amino acid solution was 2.0 mM, the concentration of L-glutamic acid solution was 0.4 mM, and the reaction was carried out at 30 ° C. with pH 5.5 (0.1 M sodium acetate buffer).

[Table 6]

When the sensor was stored at 3 ° C. in 0.02 M phosphate buffer and the response to sodium L-glutamate solution was measured as above for elapsed days, the initial response was maintained for 1 month. The immobilized enzyme membrane is thus stable for at least 1 month at 3 ° C.

Example C2

The immobilized cellophane membrane of L-glutamic acid oxidase obtained as in Example C1 was cut in a circle. It mounted on the electrode of a hydrogen peroxide electrode (made by Ishikawa Seisakusho Co., Ltd.), and coat | covered the immobilized enzyme film | membrane again on the cellulose dialysis membrane, and produced the L- glutamic acid sensor which has the same structure as the sensor structure manufactured in Example C1. Using this sensor, an L-glutamic acid assay device, such as the device of Example C1, was prepared.

A sodium L-glutamate standard solution was injected into the apparatus to measure the current value, where the correlation between the sodium L- glutamate concentration and the current value was obtained as in FIG.

Example C3

Commercial soy sauce was diluted 10-fold with 0.1 M phosphate buffer (pH 7.0) to bubble the air to prepare a sample solution in which dissolved oxygen was saturated. 1 ml of the sample solution was injected into 49 ml of 0.1 M phosphate buffer (pH 7.0), and the concentration of L-glutamic acid in the liver was calculated by the apparatus prepared in Example C1. This value was automatically analyzed by L-glutamic acid. The results were in good agreement with the quantitative values obtained by the instrument (manufactured by Technicon Instruments).

In addition, recovery tests were carried out on commercial soy sauce using the apparatuses of Examples C1 and C2 using a predetermined amount (5-100 mM) of sodium L-glutamate. The amount of sodium L-glutamate recovered by the calibration curve for each device was calculated. This yield was substantially proportional to the amount of sodium L-glutamate added as shown in FIG. 23 (apparatus of Example C1) and FIG. 24 (apparatus of Example C2).

Claims (7)

A kit for analyzing L-glutamic acid, comprising L-glutamic acid oxidase having the following characteristics and a reagent for detecting a reaction by the enzyme. A. Substrate Specificity It has a high substrate specificity for L-glutamic acid and does not act on amino acids other than L-glutamic acid. B. Optimum pH The optimum pH is around pH 7-8.5. C. Stable pH Range It is stable in the range of pH 5.5-10.5 on 37 degreeC and 60 minute hold conditions. D. Thermal Stability It is stable to 65 degreeC on the conditions of pH 5.5 holding | maintenance for 15 minutes. E. Molecular Weight The molecular weight measured by the gel filtration method (Sepadex G-200 (made by Parmacia Fine Chemicals)) is 135,000 +/- 10,000. The kit for the analysis of L-glutamic acid according to claim 1, wherein the L-glutamic acid oxidase is an enzyme having stability which does not decrease activity up to 65 ° C under a pH of 5.5 and a holding condition for 15 minutes. The kit for analyzing L-glutamic acid according to claim 1 or 2, wherein the reagent for detecting the reaction is a reagent for detecting hydrogen peroxide, ammonia or α-ketoglutaric acid. The kit for the analysis of L-glutamic acid according to claim 3, wherein the reaction detecting reagent is a single color developing agent or a combination reagent of the color developing agent and the coupler. A biosensor comprising a transducer unit which detects chemical or physical changes in an analytical sample and converts it into an electrical signal by the action of the enzyme as a receptor portion of a detected substance, wherein the enzyme is L-glutamic acid oxidase having the following characteristics. Biosensor, characterized in that using the. A. Substrate Specificity It has a high substrate specificity for L-glutamic acid and does not act on amino acids other than L-glutamic acid. B. Optimum pH The optimum pH is around pH 7-8.5. C. Stable pH Range It is stable in the range of pH 5.5-10.5 on 37 degreeC and 60 minute hold conditions. D. Thermal Stability It is stable to 65 degreeC on the conditions of pH 5.5 holding | maintenance for 15 minutes. E. Molecular Weight The molecular weight measured by the gel filtration method (Sepadex G-200 (made by Parmacia Fine Chemicals)) is 135,000 +/- 10,000. The biosensor according to claim 5, wherein the transducer is an electrode which detects a chemical change in an analytical sample and converts it into an electrical signal. The biosensor according to claim 5 or 6, wherein the receptor portion is made of an immobilized enzyme.

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