JPS6210159B2 - - Google Patents

Description

[Detailed description of the invention]

[] BACKGROUND OF THE INVENTION TECHNICAL FIELD The present invention relates to a method for analyzing L-glutamic acid in an analytical sample using a novel L-glutamate oxidase. The present invention also relates to a reagent for analyzing L-glutamic acid containing L-glutamate oxidase. Furthermore, the present invention relates to a kit for analyzing L-glutamic acid using L-glutamic acid oxidase. Prior Art Conventionally, chromatography, microbial quantification, electrophoresis, and enzyme methods have been known as methods for analyzing L-glutamic acid, and generally the chromatography method and enzyme method are used. As a chromatography method, a method using an automatic amino acid analyzer is generally used. Although this method is an excellent method with high accuracy and reliability, there are problems such as the equipment is expensive and some samples require protein removal, making the sample preparation method complicated. As enzymatic methods, methods using L-glutamic acid decarboxylase and methods using L-glutamic acid dehydrogenase are known. However, methods using these known enzymes have the following problems. That is, in the method using L-glutamic acid decarboxylase, the reaction is measured by detecting carbon dioxide gas, which is a reaction product. A method using an analyzer is adopted. The method using a Warburg manometer (b) has high accuracy, but requires considerable skill, takes a long time to measure, and has low sample processing capacity. In addition, the method using the autoanalyzer of ○Ro is a method in which carbon dioxide gas is absorbed into a sodium carbonate solution of phenolphthalein and the amount of carbon dioxide gas generated is measured from the degree of color reduction. Preliminary operations such as deaeration of carbon dioxide and oxygen while cooling are required, and gas-liquid separation is required after the reaction, making the apparatus complicated. As glutamic acid decarboxylase, enzymes derived from pumpkin or Escherichia coli are generally used;
It has urease activity, and when using a sample containing urea, there is a risk of measurement errors due to carbon dioxide derived from urea, and this enzyme is inhibited by organic acids such as acetic acid. However, it has the disadvantage that enzyme activity is inhibited in samples containing a large amount of organic acids, making it difficult to obtain accurate results. Furthermore, since the enzyme derived from E. coli exhibits activity against L-arginine and L-glutamine, accurate results cannot be obtained when a sample containing a large amount of these amino acids is used. Furthermore, the shelf life of the enzyme itself is not good. L-glutamic acid dehydrogenase reacts L-glutamic acid and water in the presence of NAD (oxidized nicotinamide adenine dinucleotide) to form α
- It catalyzes the reaction that produces ketoglutarate, ammonia, and NADH (reduced nicotinamide adenine dinucleotide), but in the method using this enzyme, the measurement of the reaction is based on the amount of NADH produced.
This is done by detecting the increase in absorbance at 340 nm. However, the equilibrium of this enzymatic reaction is tilted toward the formation of L-glutamic acid, and in order to analyze L-glutamic acid using this enzyme,
The equilibrium of the reaction must be shifted to the side that produces α-ketoglutaric acid, which requires various measures. For this purpose, a scavenger for α-ketoglutaric acid is usually added to the reaction system, but the reaction may be inhibited if the concentration is not strictly controlled. moreover
NAD concentration must also be tightly regulated. Note that when using a sample containing a substance that exhibits absorption at the measurement wavelength, such as soy sauce, the value obtained by the reaction must be corrected using the blank test value. Furthermore, since the enzyme used may have lactate dehydrogenase activity, the influence of such enzyme activity must also be taken into consideration. Recently, L-amino acid oxidase with high substrate specificity for L-glutamic acid has been developed in microorganisms belonging to the genus Streptomyces (hereinafter sometimes abbreviated as "S."), specifically Streptomyces violets. It was discovered that it can be produced by culturing Sense (see Japanese Patent Application Laid-open No. 43685/1985). The physical and chemical properties of this glutamate oxidase (hereinafter sometimes abbreviated as "known enzyme") as a protein are not clear, but the enzymatic properties described are as follows. (1) Substrate specificity When the activity towards L-glutamic acid is taken as 100, it shows a relative activity of 8.4 towards L-glutamic acid and 6.8 towards L-histidine, with no substantial activity towards other amino acids. Not shown. (2) Optimal PH PH5-6 (3) PH Stability Stable in the range of PH3.5-6.5 (held at 37°C for 1 hour). (4) Temperature stability Stable up to 50℃ (held for 10 minutes). (5) Effects of inhibitors Almost completely inhibited by mercury ions, copper ions, and diethyldithiocarbamate. There are various problems when using the above-mentioned known enzymes for the analysis of L-glutamic acid. In other words, although the known enzymes have higher substrate specificity for L-glutamic acid than conventionally known L-amino acid oxidases, they still show distinct activities for other amino acids as mentioned above, and the presence of these amino acids It cannot be used for specific quantification of L-glutamic acid. Furthermore, known enzymes do not have high pH and thermal stability, and are not necessarily considered to have good storage stability or use stability as analytical reagents. Furthermore, if copper ions are present in the sample to be analyzed, known enzyme activities are likely to be significantly inhibited, making analysis difficult. In addition, in various biochemical analyses, particularly in the analysis of enzyme activity in blood, the pH of the reaction solution is generally around neutral, but known enzymes can be completely processed at 37℃ for 1 hour at pH 7.5. It is difficult to perform analysis near neutrality because it is deactivated. Incidentally, a method for analyzing L-amino acids using L-amino acid oxidase has been known in the past, but known L-amino acid oxidases do not easily act on L-glutamic acid, so it is difficult to use such an enzyme. The method did not allow specific analysis of L-glutamic acid. [] Summary of the Invention The present invention was made to solve the problems of the prior art as described above, and was developed by the present inventors from a culture of a newly isolated microorganism of the genus Streptomyces. The newly discovered L-glutamate oxidase acts extremely specifically on L-glutamate under appropriate conditions, quantitatively consuming oxygen and producing hydrogen peroxide, ammonia, and α-ketoglutarate. As a result of repeated research on the application of this enzyme based on this knowledge, the present invention was completed. The present invention allows L-glutamate oxidase (hereinafter sometimes abbreviated as "the enzyme of the present invention") having the following characteristics to act on L-glutamic acid in an analysis sample in the presence of oxygen and water, The present invention provides a method for analyzing L-glutamic acid, which is characterized by detecting the consumption of oxygen or the production of hydrogen peroxide, ammonia, or α-ketoglutaric acid during the reaction. B. Substrate specificity The substrate specificity for L-glutamic acid is extremely high, and it does not substantially act on amino acids other than L-glutamic acid. (b) Optimal PH The optimal PH is around PH7 to 8.5. C. Stable PH range Under the conditions of holding at 37°C for 60 minutes, it is stable in the PH range of 5.5 to 10.5. D. Thermal stability: 65℃ under conditions of PH5.5 and 15 minute hold.
It is stable up to 50°C and shows about 50% residual activity at 85°C, and under the conditions of pH 7.5 and held for 15 minutes, it is stable up to 50°C and shows about 60% residual activity at 75°C. Molecular weight The molecular weight measured by gel filtration method (Sephadex G-200 (manufactured by Pharmacia Fine Chemicals)) is 135,000±10,000. The present invention also provides a reagent for analyzing L-glutamic acid, which contains the L-glutamate oxidase. Furthermore, the present invention provides a kit for analyzing L-glutamic acid, which comprises the L-glutamate oxidase and a reagent for detecting a reaction by the enzyme. Effects The enzyme of the present invention specifically acts on L-glutamic acid and does not substantially act on other amino acids.
It is the first enzyme discovered as an amino acid oxidase. Therefore, even if the sample contains many types of amino acids, the reaction of the enzyme of the present invention can be detected by employing the L-glutamic acid analysis method, analytical reagent, and analytical kit that use the enzyme of the present invention. By this, L-glutamic acid in the sample can be specifically analyzed. In addition, the L-glutamic acid analysis method, analytical reagent, and analytical kit of the present invention use the enzyme of the present invention, which has extremely high substrate specificity for L-glutamic acid.
There is no need for any pretreatment such as separating amino acids in the sample during analysis. For example, the L-glutamic acid content is an important indicator for quality evaluation of foods such as soy sauce and other liquid seasonings that contain many types of amino acids, but according to the method of the present invention, the L-glutamic acid content in these samples - Glutamic acid can be easily, rapidly, and specifically quantified. In addition, the activities of glutamate/oxaloacetate transaminase (GOT), glutamate/pyruvate transaminase (GPT), and γ-glutamyl transpeptidase (γ-GTP), which are important measurement items in clinical tests, can be measured based on the reactions of these enzymes. This can be done by directly analyzing the product L-glutamic acid using the method of the present invention, opening the way to more rapid and easy measurements of these enzyme activities. In addition, in the method of the present invention, since the enzymatic reaction type of the enzyme used is the oxidase reaction, which is most widely put into practical use in clinical tests, food analysis, etc., the detection of the enzyme reaction is also commonly used as a means of detecting the oxidase reaction. This method has the advantage that known methods can be used as they are. Furthermore, since the enzyme of the present invention has high stability compared to known enzymes and general analytical enzymes, the L-glutamic acid analysis method and analytical reagent of the present invention using such enzymes The kit for analysis is not only stable during use and storage, and has high durability, versatility, and economical efficiency, but also uses the enzyme of the present invention as an enzyme electrode.
- When applied to continuous analysis of glutamic acid, there is a significant difference in durability and stability compared to when using known enzymes. In addition, the L-glutamic acid analysis method, analytical reagent, and analytical kit of the present invention, which use the enzyme of the present invention having an optimal pH range near neutrality, have the characteristic of enzyme analysis that analysis can be performed under mild conditions. Other enzymes that are fully available and used during analysis,
The range of choices for reagents and equipment has been greatly expanded. [] Specific description of the invention (1) Enzyme of the present invention The enzyme of the present invention has extremely high substrate specificity for L-glutamic acid, does not substantially act on amino acids other than L-glutamic acid, and is a highly stable L-glutamic acid. Any glutamate oxidase may be used, regardless of its origin or origin, and its preparation method is not limited. An enzyme obtained from a culture of a microorganism of the genus Streptomyces will be listed below as an example of the enzyme of the present invention, and the specific properties and preparation method of this exemplary enzyme will be explained. (A) Enzymological and physicochemical properties of the enzyme of the present invention The enzymatic and physicochemical properties of the purified enzyme preparation of L-glutamate oxidase produced by the method of the reference example shown later are as follows. (1) Action When the enzyme of the present invention uses L-glutamic acid as a substrate, it requires 1 mol of oxygen and 1 mol of water for 1 mol of L-glutamic acid, and 1 mol of α-ketoglutaric acid, 1 mol of ammonia, and Produces 1 mol of hydrogen peroxide. (2) Substrate specificity Table 1 shows the results of reacting purified preparations of the enzyme of the present invention with various amino acids.
The concentration of each substrate was 10mM, and the reaction was conducted at PH
7.4 (0.1M potassium phosphate buffer) and
The test was conducted at PH6.0 (0.1M acetate buffer). Enzyme activity was measured by the oxygen electrode method described below.
It was expressed as a relative value of activity against L-glutamic acid.

【table】

[Table] As shown above, the enzyme of the present invention has high substrate specificity for L-glutamic acid, and for other amino acids, it shows only a slight activity (0.6%) on L-aspartic acid at pH 7.4. - other L-amino acids including glutamine and L-histidine and D-
It shows virtually no activity at all against glutamic acid, and at pH 6.0 it shows virtually no activity at all against L-aspartic acid. In contrast to the enzymes of the present invention, the known enzymes do not show activity against L-aspartic acid (0.1% or less), but have an activity of 8.4% against L-glutamine and 6.8% against L-histidine. Both enzymes exhibit different activities and differ in substrate specificity. The Km value of the enzyme of the present invention for L-glutamic acid is 2.1×10 ^-4 M at pH 7.4.
So, the Km value for L-aspartic acid is PH
7.4 is 2.9×10 ^-2 M. (3) Measurement of titer The titer of the enzyme of the present invention was measured using an oxygen electrode method. That is, 1 ml of 0.1M potassium phosphate buffer (PH7.4) containing 10mM sodium L-glutamate was placed in an oxygen electrode cell, 10μ of enzyme solution was added, and the oxygen consumption rate was measured. 1μ per minute at 30℃
The amount of enzyme that consumes mol of oxygen was defined as 1 unit (Unit; herein abbreviated as "U"). Note that the above method cannot be used to measure activity at high reaction temperatures because the dissolved oxygen concentration in the reaction solution decreases as the temperature rises. In such cases, the MBTH method (Analytical Biochemistry (Anal.
Biochem.), 25 , 228 (1968)). That is, a reaction solution containing sodium L-glutamate, catalase, and the enzyme of the present invention is incubated at an appropriate temperature for 20 minutes, trichloroacetic acid (TCA) is added to stop the reaction, and an acetate buffer (PH5.0) is added to the reaction stop solution. ) and 3-methyl-2-
After adding benzothiazolinone hydrazone hydrochloride (MBTH) and incubating at 50°C for 30 minutes, the mixture is cooled to room temperature, the absorbance at 316 nm is measured, and the produced α-ketoglutaric acid is quantified from the calibration curve. (4) Optimal PH The optimal PH is PH7~ as shown in Figure 1.
It is around 8.5. Measurement of enzyme activity at each PH uses sodium L-glutamate as the substrate, 0.2M acetate buffer (PH3.5-6.0), 0.2M potassium phosphate buffer (PH6.0-8.5) and A 0.2M glycine-sodium chloride-sodium hydroxide buffer (PH8.5-12.0) was used and the test was carried out at 30°C. In addition, in order to compare the optimum PH of the enzyme of the present invention and the known enzyme, FIG.
Quoted from Figure 1 of the publication. ) is also shown. As is clear from FIG. 1, the enzyme of the present invention also differs from known enzymes in its optimum pH. In addition, when aspartic acid is used as a substrate, the PH range of action is narrow, and the optimal PH is 7 to 8, but it has almost no effect on L-aspartic acid at PH 6.0 or lower and PH 10.0 or higher (PH 6 less than 0.1% of the activity towards glutamate at .0). (5) PH stability After holding at 37°C for 60 minutes, 45°C for 15 minutes, and 60°C for 15 minutes at each pH of 3.5 to 11.5, measure the enzyme activity for glutamic acid at PH7.4. did. As a result, under the conditions of holding at 37℃ for 60 minutes,
It is stable in the pH range of 5.5 to 10.5 (Figure 2, solid line), and stable in the pH range of 5.5 to 9.5 even when held at 45℃ for 15 minutes (Figure 3), and 60℃ for 15 minutes. Under retention conditions, PH
It was stable in the range of 5.5 to 7.5 (Figure 4). In addition, in order to compare the PH stability of the enzyme of the present invention and the known enzyme, FIG. 2 shows the PH stability curve of the known enzyme (quoted from FIG. 2 of JP-A-57-43685 and indicated by a dotted line). ) are shown together with those of the enzyme of the present invention. As is clear from Figures 2 to 4 above,
Comparing the stable PH ranges of the enzyme of the present invention and known enzymes, the two are clearly different, with the former being more stable over a wider PH range than the latter. (6) Range of suitable temperature for action A reaction was carried out for 20 minutes using sodium L-glutamate as a substrate at each temperature from 30°C to 80°C, and the enzyme activity was measured by the MBTH method described above. As a result, the optimal temperature range for the action of the enzyme of the present invention was 30 to 60°C, and the optimal temperature for action was around 50°C (Figure 5). (7) Thermal stability PH5.5, PH at various temperatures from 40℃ to 90℃
After holding each condition for 15 minutes at PH 7.5 and PH 9.5, the enzyme activity toward glutamic acid was measured at PH 7.4. As a result, it is stable up to 65°C at pH 5.5, and shows about 50% residual activity at 85°C (Fig. 6, ▲-▲). 50℃ at PH7.5
It is stable up to 75°C and shows about 60% residual activity (Fig. 6, ●-●). It is stable up to 45°C even at pH 9.5, and shows about 50% residual activity at 70°C (Fig. 6, ■-■). In order to compare the thermostability of the enzyme of the present invention and the known enzyme, the temperature stability curve of the known enzyme (quoted from Figure 3 of JP-A No. 57-43685 and indicated by a dotted line) is compared with that of the enzyme of the present invention. It is listed together with the one. As is clear from FIG. 6, the enzyme of the present invention has higher temperature stability than known enzymes. (8) Inhibition, activation and stabilization In order to investigate the effects of various additive substances on the enzyme of the present invention, each substance shown in Table 2
The enzyme reaction was carried out in a reaction solution containing mM (PH7.4). The results are shown in Table 2.

【table】

[Table] As is clear from Table 2, the enzyme of the present invention is inhibited by about 45% by parachloromercurybenzoate, but not inhibited at all by cupric chloride and diethyldithiocarbamate. On the other hand, known enzymes are completely inhibited by cupric chloride and diethyldithiocarbamate. Therefore, both enzymes also differ in terms of their effects on inhibitors. Note that an activator and stabilizer for the enzyme of the present invention have not yet been found. (9) Ultraviolet absorption spectrum (see Figure 7) λ _nax : 273 nm, 385 nm, 465 nm Shoulder: around 290 nm, around 460 nm (10) Coenzyme The enzyme of the present invention is heat-treated or treated with trichloroacetic acid (TCA) and centrifuged. The absorption of the obtained supernatant matched that of flavin adenine dinucleotide (FAD) and activated the apoenzyme of D-amino acid oxidase, indicating that the coenzyme of the enzyme of the present invention was FAD. In addition, in thin layer chromatography
It was also identified as FAD based on the Rf value. It was estimated that 2 mol of FAD was present per 1 mol 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 is 135,000± by gel filtration using Cephadex G-200 (manufactured by Pharmacia Fine Chemicals).
It was measured at 10,000. (13) Isoelectric point When measured by isoelectric focusing using Ampholine (manufactured by LKB), pI
was 6.2. (14) Crystal structure and elemental analysis The enzyme of the present invention was not crystallized, so it was not measured. (15) Purification method The enzyme of the present invention can be purified by salting out method, isoelectric precipitation method, precipitation method with organic solvent, diatomaceous earth,
The adsorption method using activated carbon or the like, various chromatography methods, etc. can be appropriately combined. Specific examples of the purification method are as shown in Reference Examples. (B) Preparation of the enzyme of the present invention Next, a method for producing the enzyme of the present invention by culturing microorganisms will be specifically described. Microorganisms used The microorganisms used in the production of the enzyme of the present invention are:
It is a microorganism that belongs to the genus Streptomyces and has the ability to produce the enzyme of the present invention. A specific example of such a microorganism is strain X-119-6, which was isolated as a single strain from the soil of Tosho-cho, Katori-gun, Chiba Prefecture. The mycological properties of this strain are described below. A. Microscopic observation Aerial hyphae are linear and have a width of 0.9 to 1.0μ.
, indicating simple branching. The sporophyte consists of a chain of many spores, forming a spiral with 2 to 5 turns. The spores are slightly oval in shape,
Its size is 0.9 to 1.0 x 1.1 x 1.2μ, and its surface is observed to have a thorn-like structure using an electron microscope. No division of basal hyphae is observed. B. Macroscopic Observation The results of macroscopic observation of growth (cultivated at 30°C for 16 days) in various media are as follows. (1) Seuculose/nitrate agar medium Growth is poor. The basal hyphae are grayish brown and do not penetrate into the agar, while the aerial hyphae are powdery and spread thinly in a radial pattern on the agar. It is grayish-brown in color and forms gray spores. No pigment formation was observed in the medium. (2) Glucose-asparagine agar medium The growth is good. The basal hyphae are whitish-yellow and rise slightly as they invade the agar. Aerial hyphae are white and sparse, and no pigment production is observed in the medium. (3) Glycerin-asparagine agar medium The growth is good. The basal hyphae are whitish-yellow and swell as they invade the agar. No aerial mycelium was formed, and no pigment was observed in the medium. (4) Starch/inorganic salt agar medium The growth is good. The basal hyphae are whitish-yellow and swell as they invade the agar. Aerial mycelium is white, abundant, and carries gray spores. No pigment formation was observed in the medium. (5) Tyrosine agar medium The growth is good. The basal hyphae are white-yellow. Aerial mycelium is white, abundant, and carries gray spores. No pigment formation was observed in the medium. (6) Nutrient agar medium The growth is extremely good. The basal hyphae are white-yellow as they invade the agar.
Get excited. Aerial mycelia are white and no spores are observed. No pigment formation was observed in the medium. (7) Yeast/malt agar medium The growth is extremely good. The basal hyphae are whitish-yellow and swell as they invade the agar. Aerial mycelium is white, abundant, and carries gray spores. No pigment formation was observed in the medium. (8) Oatmeal agar medium The growth is extremely good. The basal hyphae are white and invade the agar, but do not rise above the medium. Aerial mycelium is white, abundant, and carries gray spores. No pigment formation was observed in the medium. C Physiological properties The growth temperature range is 8 to 40°C, with optimum around 35°C. Both tyrosine agar and peptone yeast iron agar do not produce melanin-like pigments, but slightly liquefy gelatin and hydrolyze starch. D. Assimilation of various carbon sources The usability of various carbon sources on Pridham-Godelive agar medium is shown in Table 3.

[Table] The above properties are summarized as follows. That is, the aerial hyphae are spiral-shaped, and the surface of the spores is thorn-like. When grown on a medium, it exhibits a whitish-yellow or grayish-brown color, and the aerial mycelium is white to grayish-brown, with no production of soluble pigments or melanin-like pigments. In addition, starch water decomposition is strong. Based on these results and the assimilability of carbon sources shown in Table 3, the Bergey'S Manual of Determinative Bacteriology was used.
We identified this bacterial strain according to the classification system in Determinative Bacteriology, 8th edition (1974), and found that this strain belongs to the genus Streptomyces, but no known species that fully matches the characteristics of this strain was found. First, this bacterial strain was identified as a new strain, and Streptomyces sp.
(Streptomyces sp.X-119-6). Regarding this strain, we filed an application for deposit with the Institute of Microbial Technology, Agency of Industrial Science and Technology in accordance with Ministry of International Trade and Industry Notification No. 178 of 1981, and
It was accepted on June 5, 1957, and the accession number was FERM R-
6560) has been granted. The above-mentioned bacterial strain is an example of a strain that has a high ability to produce the enzyme of the present invention, and the microorganisms used in the present invention are not limited thereto. In addition, such enzyme-producing bacteria of the present invention may be subjected to conventional microbial mutagenesis methods such as ultraviolet rays, X-rays, and γ-rays.
Any highly productive mutant strain of the enzyme of the present invention obtained by mutating the enzyme by physical treatment such as irradiation or chemical treatment with a drug such as nitrosoguanidine can be suitably used. Furthermore, the production of the enzyme of the present invention by the microorganism basically utilizes the enzyme protein synthesis function of the microorganism using the gene deoxyribonucleic acid (DNA), which carries genetic information regarding the production of the enzyme of the present invention. Therefore, such genetic DNA
Microorganisms obtained by genetic engineering techniques, such as by incorporating the vector into a suitable vector and transferring it to microorganisms of other genera by transformation, or by incorporating genetic DNA into microorganisms of other genera through cell fusion using the protoplast method. The enzyme of the present invention can also be produced by Cultivation method and conditions The method and conditions for culturing the microorganism used are not particularly limited as long as the microorganism grows well and the enzyme of the present invention is sufficiently produced. suitable. The solid medium used for solid culture is no different from that normally used. In other words, the solid medium is mainly composed of natural solid materials such as bran, defatted soybeans, rice bran, corn, rapeseed meal, wheat, rice, rice husk, etc. alone or in combination of two or more, and may be used as necessary. Nutrient sources that microorganisms can quantify, such as glucose, sucrose, arabinose, fructose, mannitol,
Inositol, soluble starch, carbon sources such as ethanol, various amino acids, peptone, soybean flour, protein hydrolysates, cornstarch liquor, meat extract, yeast extract, various ammonium salts, various nitrates, nitrogen sources such as urea, various Salts such as sodium salts, potassium salts, calcium salts, manganese salts, magnesium salts, zinc salts, iron salts, phosphates, sulfates, thiamine, riboflavin, nicotinic acid, pantothenic acid, biotin, p-aminobenzoic acid, vitamins These include a medium to which a growth element such as B ₁₂ is appropriately added, or a medium prepared by forming these into an appropriate composition, size, and shape. Such a solid medium is sterilized or denatured by a conventional method, and a seed culture is inoculated to perform solid culture. In addition, culturing methods other than those described above, such as absorbing or coating a liquid medium on a suitable carrier such as a sponge (see Japanese Patent Application Laid-Open No. 14679/1983),
Even a method of culturing by inoculating seed bacteria can be adopted as long as the microorganism used can propagate and produce the enzyme of the present invention well. The culture conditions should be selected according to the type of microorganism used, and the optimal conditions for enzyme production should be selected.
Not particularly limited. Usually, for example 20-30
It may be cultured for 5 to 15 days at ℃ and pH 5 to 7. Collection of the enzyme of the present invention The enzyme of the present invention produced by culturing the microorganism used can be extracted and separated from the culture, that is, the culture medium and/or the cultured cells, by an appropriate extraction method and used as is as a crude enzyme solution, or As described above, it is purified to the purification steps according to the purpose of use according to the usual enzyme purification method. The extraction method is not particularly limited and may be performed by a conventional method. For example, extraction from solid-state cultures is usually performed with water or buffers. Furthermore, the enzyme of the present invention in the bacterial cells can be extracted by crushing the bacterial cells and solubilizing them using a conventional method. (2) Analytical sample In the present invention, the term “analytical sample” refers to
Samples that contain or are expected to contain L-glutamic acid as a component and whose content or presence of L-glutamic acid should be analyzed, or samples that release or are known to release L-glutamic acid. A sample containing the expected reaction system, where the L-
It refers to a sample for analyzing the enzyme activity involved in the system, the content of substances converted to L-glutamic acid in the system, or the presence or absence of these substances by measuring changes in the glutamic acid content. Samples classified as above include foods (e.g., soy sauce, amino acid seasonings, various extracts, liquid seasonings such as liquid stock, sake,
These include alcohol-containing foods such as mirin, seafood paste products, extracts from solid foods such as sausages and ham, etc.), biological samples (urine, blood, etc.), and other samples. racemase, GOT,
Examples include a system containing an enzyme whose reaction product is L-glutamic acid such as GPTγ-GTP and its substrate, or a system containing the above system and one or more other conjugated enzyme systems. (3) Analysis of L-glutamic acid The L-glutamic acid analysis method of the present invention is based on an enzymatic reaction system using the enzyme of the present invention for L-glutamic acid in a sample, particularly for L-glutamic acid in a sample containing a large amount of aspartic acid. Teha
It consists of an enzyme reaction system at pH 5 to 6 and an indicator substance detection system that quantitatively or qualitatively detects the indicator substance consumed or produced along with the reaction. Enzyme reaction The enzyme reaction conditions for the analysis of L-glutamic acid are as follows. That is, the reaction PH is such that the enzyme of the present invention is not inactivated and acts sufficiently on L-glutamic acid.
It is sufficient that the detection system of the indicator substance is PH.
If the pH depends on the pH, it is preferable to set the pH by considering such conditions. Usually PH5
The enzymatic reaction is carried out in the range of ~9. Note that if 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 will have some effect on L-aspartic acid near the optimum pH. Therefore, even in such samples, L-glutamic acid can be detected by selecting conditions that act on L-glutamic acid to a sufficient extent to detect the indicator substance, but do not act on L-aspartic acid. can be specifically analyzed. As such conditions, reaction conditions of pH 5 to 6 are suitable. Further, in order to maintain the pH of the enzyme reaction within a suitable range, it is preferable to use various buffer solutions as the reaction medium. As the buffer solution, the above-mentioned PH
Any substance may be used as long as it can maintain the range, does not inhibit the enzyme of the present invention, and does not affect the detection system of the indicator substance. Examples include phosphate buffer, Tris-HCl buffer, acetate buffer, citrate buffer, and veronal buffer. The reaction temperature is not particularly limited. A person skilled in the art can easily determine the optimal temperature of the enzyme of the present invention, the stable temperature, the detection system of the indicator substance used, etc. In addition, during the reaction, the enzyme of the present invention may be a soluble enzyme, an entrapment method (lattice type, microcapsule type, etc.), a carrier binding method (covalent bonding method, ionic bonding method, physical adsorption method, etc.), a crosslinking method, etc. It is used as an immobilized enzyme immobilized by a common method. The mode of immobilization is not particularly limited ("Immobilized Enzyme", edited by Ichiro Chibata, March 20, 1975, Co., Ltd.)
Published by Kodansha, reference). For example, materials for immobilization carriers include cellulose derivatives such as cellulose (cellophane), acetyl cellulose, nitrocellulose (collodion), and carboxymethyl cellulose, starch, polysaccharides such as dextran, agarose, and chitosan, collagen, fibroin, keratin, and albumin. proteins, polyacrylamide, polyvinylpyrrolidone, polyvinyl alcohol, carrageenan, alginic acid, xanthan gum, agar, and other polymeric gels, polyvinyl chloride, polystyrene, polyethylene, polypropylene, polyurethane, polycarbonate, nylon, Teflon, and photocurable adsorption resins. , synthetic organic polymers such as ion exchange resins, and inorganic polymers such as glass, silica, ceramics, and alumina are used.
Gel, granule, powder, microcapsule,
Supports in the form of tubes, fibers, holographic containers are used. Immobilization methods include entrapping the enzyme of the present invention with a polymeric gel, etc., adsorption to a porous or ion exchange material, etc., covalent bonding to a carrier by a peptide method, alkylation method, diazotization method, etc., and glutaraldehyde. , crosslinking to a carrier using a polyfunctional crosslinking agent such as hexamethylene diisocyanate, or crosslinking between enzymes. The mode of use as an immobilized enzyme differs depending on the reaction method and/or measurement method, but for example, when reacting as an enzyme electrode, it is attached tightly to the electrode as a membrane-like immobilized enzyme, or it is directly immobilized on the electrode surface. When the reaction is carried out using a reactor system, a column type reactor with a granular immobilized enzyme column, a tube type reactor with the enzyme immobilized on the inside of the tube, or an enzyme immobilized on the inside wall of the measurement container is used. be able to. Detection System for Indicator Substances The indicator substances for analyzing L-glutamic acid by the method of the present invention are oxygen consumed by the enzymatic reaction and hydrogen peroxide, ammonia, and α-ketoglutaric acid produced. Detection of each indicator substance is performed by an arbitrary method, and the present invention is not limited to the detection method. That is, although a known detection method is usually employed, it is thought that various methods to be further developed in the future may also be employed. Note that the "indicator substance detection system" includes the following detection process for each indicator substance and a process for qualitatively or quantitatively analyzing L-glutamic acid based on the detection results. Hereinafter, methods for detecting each indicator substance will be explained. (1) Detection of oxygen The method of detecting consumed oxygen is as follows:
Warburg pressure detection method (Biochemistry Experiment Course 5 Enzyme Research Methods (Part 1) pp. 35-41, published by Tokyo Kagaku Dojin Co., Ltd., August 20, 1975) and oxygen electrode method (Oxygen measurement using electrode method, Fumiji Hagiwara) Edited by Kodansha Co., Ltd., published November 20, 1977).
In the present invention, either method may be adopted. The electrodes used in the oxygen electrode method include noble metal electrodes such as platinum, gold, and iridium as the working electrode, and silver, silver/silver chloride, saturated calomel, lead, zinc, and aluminum electrodes as the reference electrode. A composite electrode (Clarke) that is integrated so that an electrolyte such as a sodium hydroxide or potassium hydroxide solution is separated from the test solution by an oxygen-permeable electrode membrane such as a Teflon membrane, a polypropylene membrane, or a polyethylene membrane. A diaphragm oxygen electrode such as a type electrode) or a separate type electrode in which the working electrode side and the reference electrode side are connected by a salt bridge are used.
Even if the method is a polarographic method,
A galvanic cell type may be used. When measuring oxygen using an oxygen electrode, dissolved oxygen in the oxygen reaction solution may be measured by a conventional method. In addition, an enzyme electrode consisting of an oxygen electrode and an enzyme of the present invention attached to the electrode surface, such as an enzyme electrode in which an immobilized enzyme or a soluble enzyme covered with a semipermeable membrane is attached closely to the electrode membrane of a Clark type electrode, can be used. You can also use it for measurement. (2) Detection of hydrogen peroxide Electrochemical analysis, spectroscopic analysis, fluorescence analysis, chemiluminescence analysis, and other methods are known as methods for detecting generated hydrogen peroxide. Either method may be used. As a general electrochemical analysis method, a hydrogen peroxide detection type electrode having a structure similar to the above-mentioned oxygen electrode is used, and measurement is performed by the current method. For example, a method using a working electrode made of a noble metal electrode such as platinum, a reference electrode made of a silver electrode, and a Clark type hydrogen peroxide electrode made of a hydrogen peroxide permeable membrane is preferably used in the present invention. Further, it may be used as an enzyme electrode in the same way as an oxygen electrode. In addition to the above method, as an electrochemical analysis method, a hydrogen peroxide decomposition catalyst such as peroxidase or molybdate is used together with the enzyme of the present invention, and hydrogen peroxide and iodine ions are reacted.
A method of measuring the decrease in the activity of iodine ions on the electrode surface using an iodine ion electrode can also be used. Note that this method is suitably used as an enzyme electrode in which an immobilized enzyme containing the enzyme of the present invention and peroxidase or a catalyst is attached to an iodine ion electrode. Spectroscopic analysis involves reacting an oxidizable coloring agent with hydrogen peroxide in the presence of peroxidase or a substance exhibiting similar activity.
The peroxidase method involves measuring the absorbance of the color produced, in which alcohol is reacted with hydrogen peroxide in the presence of catalase, the aldehyde produced is introduced into a coloring system, and the absorbance of the produced color is measured, or the aldehyde dehydrogenase method is used. Nicotinamide adenine dinucleotide (NAD)
reduced NAD (NADH)
catalase method to measure the production of
Ti () / xylenol orange type, Ti
There are chemical methods using ()/4-(2-pyridylazo)resorcinol systems, V()/xylenol orange systems, and other methods. In the peroxidase method, the color reaction is an oxidation reaction of the color former alone or an oxidative condensation reaction of the color former and a coupler. In the former method, o-dianisidine, o-
Tolidine, o-toluidine, o-aminophenol, 2,4-dichloroindophenol, benzidine, 3,3',5,5'-tetraalkylbenzidine (3,3',5,5'-tetramethylbenzidine, etc.) , 4-methoxy-1
-naphthol, 2,2'-azinodi(3-ethylbenzothiazoline-6-sulfonic acid),
Guaiac butter (guaiacol), N-(4-antipyryl)-aniline derivatives (N-(4'-antipyryl)-2-carboxy-4-hydroxyaniline, etc.), p-hydroxyphenylacetic acid, N・N-dimethyl- p-phenylenediamine and the like can be used. In the latter method, 4-aminoantipyrine derivatives such as 4-aminoantipyrine and 4-aminoantipyrinamide, 4-aminoantipyrine derivatives,
-aminophenazone, 4-amino-N・N-
phenylenediamine derivatives such as dimethylaniline, p-phenylenediamine, 4-amino-N·N-diethyl-m-toluidine,
3-Methyl-2-benzothiazolinone hydrazone (MBTH) is used, and couplers include phenol, catechol, resorcinol, hydroquinone, cresol, guaiacol, pyrogallol, orcinol, p-
Chlorophenol, p-bromophenol,
2,4-dichlorophenol, 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-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine, N-glycyl-N-ethyl-m-toluidine, p-dimethylaminophenol, o-
aminophenol, m-aminophenol,
p-methylaminophenol, 2-chloro-
Phenolic, aniline or toluidine compounds such as 6-methylphenol, 4-chloro-3-methylphenol, 3,5-dichloro-2-hydroxybenzenesulfonic acid, or 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-sulfonic acid, 2,4-dichloro-
Naphthol compounds such as 1-naphthol, 1-hydroxy-2-naphthoic acid, 4,5-dihydroxynaphthalene-2,7-disulfonic acid, naphthylamine or its derivatives, quinoline compounds such as hydroxyquinoline, aminoquinoline, etc. are used. can do. Examples of suitable combinations of color formers and couplers include 4-aminoantipyrine and phenol, N·N-dimethylaniline, N·N
-diethylaniline or N-diethyl-
Examples include a combination with m-toluidine or a combination of 3-methyl-2-benzothiazolinone hydrazone and N·N-dimethylaniline. The present invention is not limited to the type of color former or color former and coupler, and any color former can be used as long as it can be oxidized quantitatively and exhibit a change in color tone. Further, as peroxidase, oxygen derived from horseradish or sweet potato is usually used, but it is not particularly limited as long as it exhibits peroxidase-like activity. Furthermore, it may be a catalyst that exhibits the same activity as peroxidase. The method using a coloring agent in the catalase method usually uses methanol as the alcohol, and the formaldehyde produced by the reaction with hydrogen peroxide is removed in the presence of an oxidizing agent (e.g., sodium periodate, potassium ferricyanide, ferric chloride, etc.). hydrazone (e.g. 3-methyl-2-benzothiazolinone hydrazone, 4-amino-3-hydrazino-5
-mercapto-1,2,4-triazole (AHMT), etc.), and a method in which formaldehyde is reacted with acetylacetone and ammonium salt by Hunts reaction to develop color. Furthermore, using glutathione, hydrogen peroxide-glutathione peroxidase leads to oxidized glutathione, which is reduced by glutathione reductase in the presence of NADPH,
Method for measuring the amount of NADPH oxidation (Analytical Biochemistry (Anal.
Biochem.) 76 , 184-191 (1976)), there is also a method of oxidative decolorization of indigo carmine using a copper ion-histamine system and quantitative determination from the loss of color. For fluorescence analysis, homovanillic acid is reacted with hydrogen peroxide in the presence of peroxidase to form a fluorescent 2,2'-dihydroxy-
An example of this method is to convert it into 3,3'-dimethoxybiphenyl-5,5'-diacetic acid and measure the fluorescence intensity. In a similar manner, p-hydroxyphenylacetic acid, diacetylfluorescin derivatives (diacetylfluorescin, diacetyldichlorofluorescin, etc.), etc. may be used as the fluorescent reagent instead of homovanillic acid. In addition, fluorescent substances such as scopoletin and 3,5-diacetyl-1,2-dihydroltidine are oxidized with hydrogen peroxide/peroxidase and converted to non-fluorescent substances.
There are also ways to measure the reduction in fluorescence. As a chemiluminescence analysis method, luminol is oxidized by reacting with hydrogen peroxide in the presence of peroxidase, and the amount of luminescence is measured (see JP-A-57-71399 and JP-A-57-71400). be. In the same way, instead of luminol, isoluminol, pyrogallol, bis(2.4.
6-trichlorophenyl) oxalate, etc. may be used, and hemoglobin, hematin, hemin, potassium iron() cyanide, cobalt chloride, etc. may be used as a catalyst instead of peroxidase. (3) Detection of ammonia Ammonia can be analyzed by methods such as the micro Kjeldahl method, Nessler method, indophenol method, ninhydrin method, and phenosafranine method. In addition, electrochemical analysis methods such as a method for analyzing ammonium ions using a cation-selective electrode or a method for analyzing ammonia gas using an ammonia gas electrode consisting of a glass electrode equipped with a hydrophobic gas-permeable membrane are also available. You can then analyze it. Furthermore, ammonia may be detected using an enzyme electrode in which these electrodes are combined with the enzyme of the present invention, and L-glutamic acid may be analyzed. (4) Detection of α-ketoglutaric acid α-ketoglutaric acid is 3-methyl-2-
Benzothiazolinone hydrazone (MBTH)
A method of reacting with 2,4-dinitrophenylhydrazine and measuring the absorbance of the product,
A method of reacting with o-phenylenediamine and measuring the absorbance of the product, and other known methods can be used. Analytical Reagent The analytical reagent of the present invention is a reagent containing at least the enzyme of the present invention. That is, its form is not limited, and it may be a soluble enzyme in a solution, frozen, powdered or granular form,
Furthermore, as mentioned above, immobilization that is immobilized on a film-like, gel-like, granular, powder-like, chip-like, microcapsule-like, tube-like, fibrous, holofiber-like, or container-like carrier by various methods is also possible. It may also be an enzyme. Also,
In addition to the enzyme of the present invention, buffers such as liquid or powdered phosphate buffer, Tris-HCl buffer, acetate buffer, citrate buffer, and veronal buffer, salts (sodium chloride, etc.), and sugars (sucrose) ), polyhydric alcohols (glycerol, propylene glycol, sorbitol, etc.), coenzymes (FAD, etc.), other appropriate stabilizers, surfactants, etc. may be added. When analyzing L-glutamic acid, the above-mentioned analytical reagents are used in accordance with the various detection methods described above so as to obtain the necessary enzyme activity. Alternatively, an amount corresponding to each detection method may be sealed in a container such as a reagent bottle or an ampoule in advance. Analytical Kit The analytical kit is composed of an analytical reagent containing the enzyme of the present invention described above and a detection reagent that is a means for detecting the reaction caused by the enzyme of the present invention.
The detection reagent is a reagent necessary for detecting the indicator substance described above. That is, when hydrogen peroxide is used as an indicator substance, the detection reagent is, for example, a combination of peroxidase or a peroxidase-like active substance and a coloring agent or a coloring agent and a coupler, or a combination of catalase or a catalase-like active substance, alcohol, and a coloring agent. Combinations of reagents necessary for the system or reagents necessary for the coupled enzyme system, combinations of peroxidase or peroxidase-like active substances and luminescent agents, combinations of peroxidase or peroxidase-like active substances and luminescent reagents, etc. Illustrated. Specific examples of these reagents are clear from the description of "detection of hydrogen peroxide" above. Furthermore, when ammonia or α-ketoglutaric acid is used as the indicator substance, a necessary detection reagent can be similarly combined with an analytical reagent containing the enzyme of the present invention to form an analytical kit. The analytical reagent containing the enzyme of the present invention and the detection reagent described above may be mixed together to form a single reagent, and if there are components that interfere with each other, each component may be combined in an appropriate manner. It may be divided into Further, these may be prepared as a solution or powder reagent, and furthermore, they may be contained in a suitable support such as a filter paper or film to prepare a test paper or a film for analysis. In addition to the above-mentioned combination reagents, the analytical kit of the present invention may also include a standard reagent containing a certain amount of L-glutamic acid. As a preferred example of the analytical kit of the present invention, L-
Examples include kits for analyzing glutamic acid. For example, in the case of a kit using the peroxidase method, the enzyme of the present invention is usually 0.02 units (U) or more per test, and peroxidase 1 to
10U/test, when using 4-aminoantipyrine and phenol or N/N-dimethylaniline as coloring agents, these reagents are used in an amount of 1 mol or more, preferably 2 mol or more, based on the hydrogen peroxide produced. . (4) Specific Examples Hereinafter, one specific example of the analytical method, analytical reagent, and analytical kit of the present invention will be shown as an example.
In addition, a method for producing the enzyme of the present invention will be shown as a reference example. However, the present invention is not limited to these Examples and Reference Examples. Example 1 (1) Preparation of kit Reagent A: 1 mg of L-glutamate oxidase
(10U/mg), peroxidase 5mg (100U/mg)
mg; derived from horseradish, manufactured by Toyobo Co., Ltd.) and 4
-Aminoantipyrine (40 mg) was dissolved in 2 ml of 0.2M potassium phosphate buffer (PH6.5) so as to be contained in one reagent bottle, and lyophilized using a conventional method. Reagent B: Dissolve 40 mg of phenol in 0.1 M phosphate buffer (PH6.5) and add 100 ml to a reagent bottle, or add 100 mg of dimethylaniline.
It was dissolved in 0.1M potassium phosphate buffer (PH6.5) and placed in a reagent bottle as 100ml. (2) Operation method Dispense 0.1 ml each of the standard solution of sodium L-glutamate and water into test tubes, dissolve the lyophilized product of reagent A in 100 ml of reagent B, or any solution, and perform each of the above tests. 0.9 ml was added to each tube and incubated for 20 minutes at 37°C with aerobic shaking. 500nm when using the above as a blind control with water
When using the above, the absorbance at 565 nm was measured. These calibration curves are shown in Figure 8 (Reagent B
) and FIG. 9 (when reagent B is used). Example 2 (1) Preparation of reagent Coloring reagent: 20 mg of phenol, 20 mg of 4-aminoantipyrine, and 3 mg of horseradish peroxidase (100 U/mg) were dissolved in 50 ml of 0.2 M potassium phosphate buffer (PH7.4). . L-glutamate oxidase: purified enzyme
300μg (55U/mg) was dissolved in 30ml of 0.02M potassium phosphate buffer (PH7.4) (0.55U/mg).
ml). (2) Procedure Dispense 0.8ml of coloring reagent into a test tube, add 0.1ml of L-glutamate oxidase solution, and incubate at 37°C.
It was placed in a constant temperature bath and heated for 5 minutes. Standard sodium L-glutamate solution (0-2 μmol/ml)
Or add 0.1 ml of sample solution (a solution prepared by diluting various soy sauce products 150 times with water) and stir well.
Incubate for 20 minutes with aerobic shaking at °C. Absorbance at 500 nm was measured using a blind test as a control, and a calibration curve shown in FIG. 10 was created. The quantitative value of L-glutamic acid in the sample obtained from this calibration curve was determined by the conventional method using L-glutamic acid decarboxylase (method of measuring the degree of color reduction of phenolphthalein using a Technicon autoanalyzer). The results are shown in Table 4 (shown after Example 3) in comparison with the quantitative values of L-glutamic acid. Note that the correlation coefficient γ=0.997 and the regression equation y=1.005x−0.575. Example 3 0.1M potassium phosphate buffer (PH7.4) in a test tube
0.7ml, catalase (1000U/ml; derived from beef liver,
0.1 ml of L-glutamate oxidase (0.6 U/ml) solution and 0.1 ml of L-glutamate oxidase (0.6 U/ml) solution
was heated at 37°C for 5 minutes. Standard L-
Sodium glutamate solution (0-5μmol/
ml) or a sample solution (same soy sauce dilution solution as in Example 2) was added to start the reaction. 37
After incubation with aerobic shaking for 20 minutes at ℃, the reaction was stopped by adding 0.1 ml of 25% trichloroacetic acid. Add 1M acetate buffer (PH
5.0) 1.9 ml and 0.8 ml of 0.1% 3-methyl-2-benzothiazolinone hydrazone hydrochloride solution were added, stirred, and incubated at 50°C for 30 minutes. After cooling to room temperature, the absorbance at 316 nm was measured using a blind test as a control, and a calibration curve shown in FIG. 11 was created. The quantitative value of L-glutamic acid in the sample obtained from this calibration curve was compared with the quantitative value of L-glutamic acid in the sample solution obtained by the conventional method using L-glutamic acid decarboxylase, and the results are shown in FIG. Note that the correlation coefficient γ=
0.996, and the regression equation was y=1.026x−3.122.

[Table] Example 4 (1) Preparation of reagent Color reagent: 30 mg of phenol, 30 mg of 4-aminoantipyrine, horseradish peroxidase 4
mg (100 U/mg) and 1 mg (10 U/mg) of L-glutamate oxidase were dissolved in 50 ml of 0.2 M potassium phosphate buffer (PH6.5). Substrate solution A: Sodium L-aspartate
200 mg and 15 mg of α-ketoglutaric acid were dissolved in 10 ml of 0.1M potassium phosphate buffer (PH7.0). Substrate solution B: 140 mg L-alanine and α-
15 mg of ketoglutarate was dissolved in 10 ml of 0.1M potassium phosphate buffer (PH7.0). (2) Procedure Measurement of activity of glutamate-oxaloacetate transaminase (GOT) Take 0.2 ml of substrate solution A into a test tube and add standard enzyme solution (manufactured by Boehringer Yamanouchi Co., Ltd., 380U/
After adding 0.1 ml of 25% trichloroacetic acid and incubating at 37°C for 30 minutes, the reaction was stopped by adding 0.1 ml of 25% trichloroacetic acid. Add 0.1ml and L of 1M potassium phosphate buffer (PH6.5) to the reaction stop solution.
-Coloring reagent containing glutamate oxidase
0.5 ml was added and incubated at 37°C for 20 minutes. Absorbance at 500 nm was measured using a blind test as a control, and a calibration curve shown in FIG. 12 was created. Activity measurement of glutamate/pyruvate transaminase (GOT) Take 0.2 ml of substrate solution B into a test tube and add standard enzyme solution (manufactured by Boehringer Yamanouchi Co., Ltd., 140 U/mg).
After adding 0.1 ml and incubating at 37°C for 30 minutes, 0.1 ml of 25% trichloroacetic acid was added to stop the reaction. The content of L-glutamic acid in the reaction termination solution was measured in the same manner as in , and the calibration curve shown in FIG. 13 was created. Example 5 L-glutamate oxidase (0.5U/ml)
Seal 1 ml of 0.1M acetate buffer (PH5.5) containing 30℃ water into a polarographic oxygen electrode cuvette, and add 50μ of L-glutamic acid standard solution (0 to 2μmol/ml). The oxygen consumption was measured, and a calibration curve shown in FIG. 14 was created. The quantitative values of L-glutamic acid in samples (various soy sauce products) determined from this calibration curve were compared with the quantitative values of L-glutamic acid in samples obtained by the conventional method using L-glutamic acid decarboxylase, and the results are shown in Table 5. . In addition, correlation coefficient γ = 0.953, regression formula y = 0.930X +
It was 6.11.

【table】

[Table] Example 6 L-glutamate oxidase (12.5U/ml)
Transfer 0.5 ml of the solution to a porous nitrocellulose membrane (manufactured by Toyo Kagaku Sangyo Co., Ltd., TM-5; pore size 0.1 μm, diameter 25 mm,
The L-glutamate oxidase membrane preparation was obtained by adsorption and fixation by dropping and suctioning onto a membrane (thickness: 140 μm). Cut the above immobilized enzyme membrane into a circle (diameter 5.0 mm)
After that, use a diaphragm oxygen electrode (manufactured by Ishikawa Seisakusho Co., Ltd., U-2).
type) gas permeable membrane (Teflon membrane, membrane thickness 10μm)
A cellulose dialysis membrane was further attached to the immobilized enzyme membrane to produce an L-glutamate sensor. Using the same sensor, a known concentration (0.05 to 1.0 μ
The current reduction value with respect to a standard solution of sodium L-glutamate (mol/ml) was measured, and a calibration curve shown in FIG. 15 was created. Example 7 L-glutamate oxidase (448U/ml)
0.1 ml of the solution was dropped onto a 30 mm x 30 mm square cellophane membrane (film thickness 30 μm), dried, and infiltrated with 0.1 ml of 2.5% glutaraldehyde solution, dried overnight at 4°C to immobilize L-glutamate oxidase. A cellophane membrane was obtained. The above immobilized enzyme membrane was mounted on a diaphragm oxygen electrode in the same manner as in Example 6 to produce an L-glutamic acid sensor. When the current reduction value with respect to the L-glutamic acid standard solution was measured using this sensor in the same manner as in Example 6, a linear relationship was obtained in the concentration range of 0.05 to 1.0 mM. Using the above sensor, the responsiveness of each PH to a sodium L-glutamate solution (0.8mM) was measured (30°C), and the results are shown in FIG. 1st
As is clear from Figure 6, the optimum pH of the sensor is PH
It was around 6.5-9.5. Furthermore, the responsiveness of the above sensor to various amino acids was measured, and the results are shown in Table 6. In addition, L
- Amino acids other than glutamic acid were measured using a solution with a concentration of 2.0mM, and L-glutamic acid was measured using a solution with a concentration of 0.4mM, and the measured values were converted to calculate a relative response value. The reaction was carried out at PH5.5 (0.1M acetic acid-sodium acetate buffer).

[Table] The above sensor was stored in 0.02M phosphate buffer at 3°C, and L
- When the responsiveness to a sodium glutamate solution was measured, the initial responsiveness was maintained for one month. Therefore, the immobilized enzyme membrane is stable at 3° C. for at least one month. Example 8 L-glutamate oxidase 2U and L-
0.1M acetate buffer (PH
5.5) Seal 1 ml into a Clark-type oxygen electrode cuvette circulating constant temperature water at 30°C, add 20 μ of E. coli-derived glutaminase standard solution (4 mU to 20 m
U) was injected, the oxygen consumption rate was measured, and a calibration curve shown in FIG. 17 was created. Similarly, soy sauce koji suspension (10g of soy sauce koji to 50ml)
Homogenize with 0.1M phosphate buffer (PH7.0) at 1000 rpm for 5 minutes, then add 50μ of the same buffer to make 300ml), measure the oxygen consumption rate, and determine the glutaminase activity per 1g of soy sauce koji. When I asked for it, it was 3.7U. Reference example: 20g of wheat bran and 16g of water in a 500ml Erlenmeyer flask
Streptomyces sp.
-119-6 (Feikokenbacterium No. 6560) was inoculated, and 28
A seed culture was prepared by culturing at ℃ for 7 days. 25 5-volume Erlenmeyer flasks with 200 bran each
g and 160 ml of water, autoclaved at 120℃ for 30 minutes, inoculated with the above-mentioned seed culture, and incubated at 28℃ for 2 hours.
After culturing for one day, the temperature was lowered to 20°C and the culture was continued for another two weeks. The obtained culture was immersed in 37.5 g of water for 1 hour, filtered, and further passed through diatomaceous earth to obtain a crude enzyme solution of about 3.4 g. Ammonium sulfate was added to this crude enzyme solution until 50% saturation, and the resulting precipitate was collected by centrifugation, dissolved in 0.02M acetate buffer (PH5.5) 3.9, and heated at 57°C for 30 minutes. After cooling the heat-treated enzyme solution to below 5°C, double the amount of pre-chilled ethanol was added, and the precipitate formed was collected by centrifugation.
It was dissolved in 0.02M phosphate buffer (PH7.4) 2 and dialyzed against the same buffer overnight. The precipitate generated during dialysis was removed by centrifugation, and the supernatant was equilibrated with the same buffer.
Pass the adsorbed enzyme through a DEAE (diethylaminoethyl)-cellulose column (3.5 x 50 cm) and remove it from sodium chloride.
Elution was performed using the same buffer containing 0.35M. Collect the eluted active fraction and contain 0.05M salt.
Dialysis was performed overnight against 0.05M acetate buffer (PH5.5). This dialyzed solution was passed through a DEAE-Sepharose CL-6B (manufactured by Pharmacia Fine Chemicals) column (2 x 10 cm) equilibrated with the same buffer, and the adsorbed enzyme was removed using a linear gradient method with 0.05 to 0.75 M of sodium chloride. It eluted. The discharged active fraction was collected, and after dialysis and concentration, it was applied to a Cephadex G-200 (manufactured by Pharmacia Fine Chemicals) column (2.5 x 120
After gel filtration is performed using 0.02M potassium phosphate buffer (PH
7.4). This dialysate solution was centrifuged, the supernatant liquid was microfiltered, and then freeze-dried to obtain a purified specimen of L-glutamate oxidase (specific activity: 55.1 U/mg).
30 mg of protein (yield 18.4%) was obtained.

[Brief explanation of the drawing]

FIG. 1 shows the action PH range of the enzyme of the present invention (solid line) and the known enzyme (dotted line). Figure 2 shows the stable PH range (37
°C, held for 60 minutes). Figure 3 shows the stable PH range (held at 45°C for 15 minutes) of the enzyme of the present invention. Figure 4 shows the stable PH range (60°C, 15 minutes) of the enzyme of the present invention. FIG. 5 shows the optimal temperature range for the action of the enzyme of the present invention.
Figure 6 shows the enzyme of the present invention (solid line) and the known enzyme (dotted line).
〇……〇) Indicates the stable temperature range. In Figure 6, ▲-▲ is PH5.5, ●-● is PH7.5, ■-■ is PH
The temperature stability curves under each condition of 9.5 are shown. 7th
The figure shows the ultraviolet absorption spectrum of the enzyme of the present invention.
8 and 9 show the calibration curves of L-glutamic acid when a phenol solution or a dimethylaniline solution was used as reagent B in the kit of Example 1, respectively. FIG. 10 shows a calibration curve for L-glutamic acid in Example 2. FIG. 11 shows a calibration curve for L-glutamic acid in Example 3. Figure 12 shows the results in Example 4.
A calibration curve of GOT activity is shown. Figure 13 shows Example 4
The standard curve of GPT activity is shown in FIG. FIG. 14 shows a calibration curve for L-glutamic acid in Example 5. FIG. 15 shows a calibration curve for L-glutamic acid obtained using the enzyme electrode of Example 6. 16th
The figure shows the responsiveness of the enzyme electrode of Example 7 at each pH. FIG. 17 shows a calibration curve for glutaminase activity in Example 8.

Claims (1)

[Claims] 1. L-glutamic acid oxidase having the following characteristics is allowed to act on L-glutamic acid in an analysis sample in the presence of oxygen and water, and the consumption of oxygen accompanying the reaction or hydrogen peroxide, ammonia or A method for analyzing L-glutamic acid, which comprises detecting the production of α-ketoglutaric acid. B. Substrate specificity The substrate specificity for L-glutamic acid is extremely high, and it does not substantially act on amino acids other than L-glutamic acid. (b) Optimal PH The optimal PH is around PH7 to 8.5. C. Stable PH range Under the conditions of holding at 37°C for 60 minutes, it is stable in the PH range of 5.5 to 10.5. D. Thermal stability: 65℃ under conditions of PH5.5 and 15 minute hold.
It is stable up to 50°C and shows about 50% residual activity at 85°C, and under the conditions of pH 7.5 and held for 15 minutes, it is stable up to 50°C and shows about 60% residual activity at 75°C. Molecular weight The molecular weight measured by gel filtration method (Sephadex G-200 (manufactured by Pharmacia Fine Chemicals)) is 135,000±10,000. 2 L-glutamic acid in an analysis sample containing L-aspartic acid together with L-glutamic acid
- The analytical method according to claim 1, wherein the analysis using glutamate oxidase is performed under conditions of pH 5 to 6. 3. A reagent for analyzing L-glutamic acid containing L-glutamic acid oxidase having the following properties. B. Substrate specificity The substrate specificity for L-glutamic acid is extremely high, and it does not substantially act on amino acids other than L-glutamic acid. (b) Optimal PH The optimal PH is around PH7 to 8.5. C. Stable PH range Under the conditions of holding at 37°C for 60 minutes, it is stable in the PH range of 5.5 to 10.5. D. Thermal stability: 65℃ under conditions of PH5.5 and 15 minute hold.
It is stable up to 50°C and shows about 50% residual activity at 85°C, and under the conditions of pH 7.5 and held for 15 minutes, it is stable up to 50°C and shows about 60% residual activity at 75°C. Molecular weight The molecular weight measured by gel filtration method (Sephadex G-200 (manufactured by Pharmacia Fine Chemicals)) is 135,000±10,000. 4. The analytical reagent according to claim 3, which contains L-glutamate oxidase and a buffer suitable for the reaction of the enzyme. 5. The analytical reagent according to claim 3 or 4, wherein the L-glutamate oxidase is an immobilized enzyme immobilized on a carrier. 6. 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. B. Substrate specificity The substrate specificity for L-glutamic acid is extremely high, and it does not substantially act on amino acids other than L-glutamic acid. (b) Optimal PH The optimal PH is around PH7 to 8.5. C. Stable PH range Under the conditions of holding at 37°C for 60 minutes, it is stable in the PH range of 5.5 to 10.5. D. Thermal stability: 65℃ under conditions of PH5.5 and 15 minute hold.
It is stable up to 50°C and shows about 50% residual activity at 85°C, and under the conditions of pH 7.5 and held for 15 minutes, it is stable up to 50°C and shows about 60% residual activity at 75°C. Molecular weight The molecular weight measured by gel filtration method (Sephadex G-200 (manufactured by Pharmacia Fine Chemicals)) is 135,000±10,000. 7. The analytical kit according to claim 6, wherein the reaction detection reagent is a reagent for detecting hydrogen peroxide, ammonia, or α-ketoglutaric acid. 8. The analytical kit according to claim 6 or 7, wherein the reaction detection reagent is a single color former or a combination reagent of a color former and a coupler.