JPS5942896A - Method, reagent and kit for analysis of l-glutamic acid - Google Patents

Abstract

PURPOSE:To determine the amount of L-glutamic acid in a specimen in high accuracy, by using a novel glutamic acid oxidase having extremely high substrate specificity to L-glutamic acid. CONSTITUTION:An L-glutamic acid oxidase having extremely high substrate specificity to L-glutamic acid, essentially inactive to amino acids other than L-glutamic acid, and having high stability to keep its activity at 5.5pH and 65 deg.C for 15min, e.g. the L-glutamic acid oxidase produced by Streptomyces sp. X-119-6 (FERM-P No.6560), is made to react with the L-glutamic acid in a specimen in the presence of oxygen and water, and the amount of the L-glutamic acid can be measured by determining the amount of oxygen consumed by the reaction, or the amount of hydrogen peroxide, ammonia or alpha-ketoglutaric acid produced by the reaction.

Description

DETAILED DESCRIPTION OF THE INVENTION [1] 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 monoglutamic acid okindase. The present invention also relates to a reagent for analyzing monoglutamic acid containing L 2 glutamate oxidase. Furthermore, the present invention relates to a method for preparing monoglutamic acid using L-glutamic acid oquinodase.

PRIOR ART Conventionally, the chroma 1-graph method, the microbial foot method, the electrophoresis method, and the enzymatic method have been known as methods for analyzing L-glutamic acid, but in general, the chroma-1 graph method and the enzymatic method are known. is used.

As the chroma I/graph method, a method using an automatic amino acid analyzer is generally used. Although this method is an excellent method with high accuracy and reliability, it has problems in that the equipment is expensive, protein removal is required depending on the sample, and the sample preparation method is complicated.

As for the enzymatic method, ``L-glutamic acid decarboxylase/Jt'' and ``L-glutamic acid dehydrogenase are known as methods A1. However, the methods using these known enzymes may have the following problems: - In the method using glutamic acid decarboxylase, the reaction is measured by detecting carbon dioxide, which is the reaction product. Usually, the method using a Warburg manometer or the force method using an autoanalyzer is adopted. The method (2) using a Warburg manometer has high accuracy, but requires considerable skill, requires a long time for measurement, and has low sample processing capacity. In addition, in the case of @1 using an ary feeder, the carbon dioxide gas is phenol-tutaray. Absorb in sodium carbonate solution and reduce [<! Since this method measures the carbon dioxide gas generated from Maro, it is safe to carry out preliminary operations such as degassing the carbon dioxide and oxygen in the enzyme solution and buffer solution while cooling them, and the gas-liquid separation is performed after the reaction. However, there is a problem in that the equipment is complicated. As the glutamic acid decarboxylase, Kapocha or Escherichia coli 111 enzyme is generally used.
The enzyme derived from Kabodya has urease activity,
When using samples that contain urea, there is a risk of measurement errors due to carbon dioxide derived from urea.Also, this enzyme is inhibited by organic acids such as acetic acid, so samples containing a large amount of organic acids may However, since the enzyme derived from E. coli is active against L-arginine and L-glutamine, it is difficult to target samples containing a large amount of these amino acids. In the case of 11; it is not possible to obtain a reliable 'LQ+'i fruit.Also, the preservation of the enzyme itself is good<t,1.

■I7-Glutamic acid deoxygenation hydrogenase L Reacts glutamic acid and water in the presence of NAD (oxidized form-: J-f-amino-1-no-denine dinucleotide)/7-Ke1-glutaric acid, In a method that catalyzes a reaction that produces ammonia and NADTI (reduced nicodinamide dinucleotide), or uses this enzyme,
Measuring reactions ii41.1. This is done by detecting the increase in absorbance at 340 nm. ,−
In order to analyze glutamic acid, the equilibrium of the reaction must be shifted to the side that produces α-ketoglutaric acid, and for this reason various dihydrogens are required. For this purpose, a scavenger for σ-ketoglutaric acid is usually added to the reaction system, but the reaction may be inhibited if the concentration is not strictly controlled. Furthermore, the NAD/A degree must be strictly controlled.

In addition, when using a sample f1 that contains a substance that exhibits absorption at the measurement wavelength such as Habaura T and L, the value obtained by reaction must be corrected using the blank test value.

Furthermore, the influence of lactate dehydrogenase activity in the enzyme used must also be taken into account.

Recently, it has been shown that amino acid oxidase has high substrate specificity for -glutamic acid,
reptomyces; sometimes abbreviated as ISl. ) was found to be produced by culturing microorganisms belonging to the genus S1-Leftomyces violetscens (see JP-A-57-48685 Yumiko M). The physical and chemical properties of this glutamic acid 'A-1 notase (hereinafter sometimes abbreviated as "known enzyme") as a protein are not clear. The enzymatic properties of Squid X are as follows.

(1) Substrate specificity L If the relative activity is 100 for L-glutamic acid and 8.4 for L-glutamine and 6.8 for L-histidine, then the relative activity for other amino acids is Substantive activity is small against.

(2) Optimum pH pH5-6 (81pH stability 1) H8.5-6.5 range (held at 37°C for 1 hour)
It is stable.

(4) Temperature stability Stable up to 50°C (held for 10 minutes).

(5) Effects of inhibitors Mercury ions, copper ions, and 1:hi-diethyldithiob J Rubamei I are almost completely inhibited.

There are various problems when using known enzymes for the analysis of L-glutamic acid as described in Section 1. Namely, compared to the conventionally known L-amino acid oxidase, the known enzymes have a lower Although heterosexuality is high,
As mentioned above, since other amino acids still have no clear activity, I
, cannot be used for specific quantification of curtamic acid. Furthermore, known enzymes do not have high stability, and their storage stability and usage stability as analytical reagents are not considered to be necessarily good. Furthermore, when copper ions and ions are present in the analysis sample, known enzyme activities are significantly inhibited, making analysis difficult.1. (
I thought so and did A.

Conventionally, a method for analyzing amino acids using I-amino acid oxidase has been known1), but the known [-amino acid oxidase does not act on L-glutamic acid, but unlike this I-amino acid oxidase. In the method using enzymes, L-
Specific analysis of glutamic acid was not possible.

(1) Summary of the Invention The present invention has been made to solve the problems of the prior art as described above, and it is possible to improve The novel L-glutamate oxodase discovered by the inventors acts extremely specifically on L-glutamate under appropriate conditions, and periodically consumes oxygen to produce hydrogen peroxide, ammonia, and α-ketoglutarate. Based on this knowledge, we conducted research on the application of the enzyme of the present invention, and as a result, we completed the present invention.

The present invention provides 1. monoglutamic acid in an analysis sample.
No1 for glutamic acid (τ1 special 1, 1.j, f
It has very high Il (J), has no substantial effect on amino acids other than L-glutamic acid, and has a low stability ratio of 1-glutamic acid oxidase (hereinafter abbreviated as the enzyme of the present invention 1). In the presence of oxygen and water, the reaction is characterized by the consumption of oxygen, hydrogen peroxide, ammonia or ketoglutarate, and the analysis of glutamic acid. This is provided by A.

Furthermore, the present invention also provides a monochromator [1
When the aforementioned monoglutamic acid okindase was allowed to act on L-glutamic acid 112 in an analytical sample containing aspartic acid of 1 to 1 in the presence of acid A and water, pH 5.
We present a method for analyzing monoglutamic acid, which is characterized by detecting the consumption of oxygen or the production of hydrogen peroxide, ammonia or ketoglutaric acid, which are involved in the reaction. .

Further, the present invention provides a reagent for analyzing L glutamic acid, which is formed by niJ, L = glutamic acid oquindase (T).

Furthermore, the present invention provides a kit for analyzing monoglutamic acid, which comprises (7) glutamate oxidase and a reagent for detecting a reaction by the enzyme.

Effect The enzyme of the present invention specifically targets L-glutamic acid,
It is virtually incompatible with other amino acids and is the first enzyme discovered as an amino acid oxidase.

Shitakatte, WIIIMIWIi II II @ 11
11 @M-class]y Even if the sample contains amino acids, L-glutamic acid in the sample can be specifically analyzed by allowing the enzyme of the present invention to act and detecting the reaction. Furthermore, since the specificity for L-glutamic acid is extremely high, no pretreatment such as separating amino acids in the sample is required for analysis.

For example, 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, and according to the method of the present invention,
monoglutamic acid in these samples easily and quickly.
Moreover, you can do something like C, which has a specific value of 1 to 1.

In addition, glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (G
The activity measurement of P i'') can be carried out by directly analyzing [, -glutamic acid, a reaction product of the cholera enzyme θ, by the method of the present invention, which makes it possible to measure the activity of these enzymes more quickly and I could easily get to 11.

Furthermore, since the enzymatic reaction of the enzyme of the present invention is the oxidase reaction that is most widely put into practical use in clinical testing or food analysis,91 the detection of the enzyme reaction can also be used as a means of detecting the oxidase reaction. There is an advantage that a widely used known method can be used as is.

Furthermore, the enzyme of the present invention has high stability compared to known enzymes and general enzymes for U1, so the analytical reagent of the present invention W Ill (!1! is stable during its existence and use). Not only is it highly versatile and economical, it is also advantageous when applying the enzyme of the present invention to the analysis of L-glutamic acid as an enzyme electrode.

[11] Detailed Description of the Invention 1) Enzyme of the Invention The enzyme of the invention has extremely high quality specificity for L-glutamic acid, substantially acts on amino acids other than L-glutamic acid, and has high stability. Any L-glutamate oxidase with a high level of L-glutamate oxidation is sufficient, regardless of its origin or origin, and its preparation method.As an example of the enzyme of the present invention, the specific quality and preparation method of this exemplified enzyme from Streptomyces are described below. explain.

(N 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 below are as follows.

(1) Action When the enzyme of the present invention uses L-glutamic acid as a substrate, Im
ol oxygen and 1 m01 water, 1 m01 a-ketoglutaric acid, 1 mO+ ammonia and 11110
1 of hydrogen peroxide is produced.

C00II ('00!11 CH2(',, +(:! 1 ) CH2+02to1120-CI!, 4 1- NH3+
H2O2) I CHNII2C, -0 ] I C0OII C00IIL Glutamic acid (1-Litocurtaric acid (2) Substrate 411.1,1.j Bamboo The results of allowing the purified preparation of the enzyme of the present invention to act on various amino acids were Table 1. The concentration of each substrate is 1011M.
The reaction was carried out with pl-17,4 (0.1M potassium phosphate buffer) and [)l-16,0 (0.1M acetate buffer). Enzyme activity was measured by the oxygen electrode method described below and expressed as a relative value of activity to L-glutamic acid.

Table 1 (1) Table 1 (2) As shown, the enzyme of the present invention has high substrate specificity for L-glutamic acid, and has a high substrate specificity for other amino acids at pH 7.4.
Slight activity on L-aspartic acid (0.6%
), and other L-amino acids including L-glutamic acid and L-histidine, and D-glutamic acid showed virtually no activity; It also shows virtually no activity against.

In contrast to the second enzyme of the present invention, the known enzyme is L as described above.
-Small activity against aspartic acid (0.1%)
(below) is an enzyme that exhibits 8.4% activity against L-glutamino and 6.8% activity against L-histidine, and both differ in substrate property yI:.

The b value of the enzyme of the present invention for rutamic acid is p
2.1 x 10'M in I-1,7,4 and the km value for L-aspartic acid is 1) 2.9 x 10'M in I-17,4.

(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 0.1 M potassium phosphate buffer (pH 7.4) containing 10 mM monoglutamate.
into the oxygen electrode cell and 101I7? The enzyme solution was added to measure the oxygen consumption rate. 17 per minute at 30°C
Zrnol oxygen consuming enzyme 17” was 1 medium (1
nitH is abbreviated as r U J in this specification. ).

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 MBT I'l method (Analytical Biochemistry (, 4na1.13
iochem,'), 25. 2'28 (,19
68)). 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 the reaction solution is incubated with acetate buffer. After adding solution (pH 5,0) and 3-methyl-2-benzothiazot hydrazone hydrochloride (MBTH) and incubating at 50°C for 30 minutes, the temperature was lowered to room temperature, and the absorbance at 3160 rn was measured. The amount of α-ketoglutaric acid produced (7) is determined from the line.

(4) Optimum p tt Optimum p I-1 is as shown in FIG. 1, I) H7-8.
It is around 5. Measurement of enzyme activity at each pH was performed using L~sodium glutamate as the substrate and buffered i
0.2M oxacic acid buffer (1) H8,5-6.0 as tk
), 0, 2 M potassium phosphate buffer i1k (1) H6
, O~8,5) and 0.2 M glycine-puttrium chloride-sodium hydroxide buffer (1) H8,5~12.
0) at 30°C.

In addition, the number in Figure 1 shows the optimum 1)H of the enzyme of the present invention and the known enzyme.
For comparison, the enzyme of the present invention (solid line) and the known enzyme (dotted line:
Quoted from Figure 1 of Japanese Patent Application Laid-open No. 57-48685.

) The pH activity curve of (71) is shown.

As is clear from FIG. 1, the enzyme of the present invention also differs from known enzymes in optimum 1)H.

In addition, the working pH when aspartic acid is used as
The range is narrow, the optimum pH is 7-8 or pt+6.0
In the above and pi-ito, o and 2, (+) TI6. which has almost no effect on L-aspartic acid.
1 to glutamic acid at 0, 0.1% of rJ
below).

(51 pH stability 1) H8, 5 to 11.5 each 1) I-1 3 degrees
G, 60 minutes, 45°C, 115 minutes, 60'C, 1
After holding each condition for 5 minutes, the enzyme activity toward glutamic acid was measured in I) H7,4.

As a result, under the conditions of holding at 37°C for 60 minutes, I) f15
．． It is stable in the range of 5 to 10.5 (Fig. 2).

solid line), pH 5.5 even under the condition of holding at 45°C for 115 minutes.
Stable in the range of ~9.5 [Yes (Figure 3), 6
Condition of holding at 0°C for 915 minutes f'l -+? l;1.1
+II 5.5-7.5 1ii', i 11111+
'', I-(7 Stable Horns: (Figure 4).

/, (Fig. 2 shows the pI-1 of the enzyme of the present invention and the known enzyme.
Stable? 11 of the Jt axis, the known enzyme Toan'jji
The curve (quoted from FIG. 2 of JP-A-57-48685, with the dotted line smaller) is marked as that of the enzyme of the present invention. .

As is clear from Figures 2 to 4 of ``C5+,',S,+jiJF? 5 is more stable in a wider pH range than before.

(61 action 21 & range of vortex 308C-il (+'CO) t5 fn! +g
tc to Ic L-Rirshi 9 Minic acid triad M B TI+ /7: TEAY 24 Measurement of lfi property jj -).

That hook 14! The application range of f′1 river for wood grain enzyme is 30~60°C, and the suitable temperature for action is 50°C (
-3 near one] (Figure 5).

(7) Thermal stability pli at various temperatures from 40°C to 90°C 5. 5
, I) H7,5 and J: and pH9.5 monograph A11te1
After holding for 5 minutes, pH 7. 4, the enzyme activity against glutamic acid was measured.

As a result, pH 5. + in 5. l G 5°C
J: There is a fixed amount, about 50% remaining at 856C (f/ll
IIII is smaller (No. (:Figure, ^-^,). pl-17.
Stable up to IJ fi 110c at 5, 75
About 60% of the residual activity was reduced to 1-(Fig. 6, 9-') at
opH9. 5 to 1,; even if it is 45° and 1 te stable J) ri, 70°C te about 5 o < 6 1/J remaining (
f/Jr 11wo-+zu (Fig. 6. #-n),.

In addition, in order to compare the thermostability +'l of the enzyme of the present invention and the known enzyme, the temperature stability curve of the known enzyme ('lr'?
4 a (l s 5 +: Quoted from Figure 3 of the publication 1,
, ! ,',I got it on the i-line. ) of the enzyme of the present invention.
J = III 1-/2.

As is clear from FIG. 6, the present IC: Ming enzyme has higher temperature stability than known enzymes.

(8) Various 14 additives to inhibition, activation and stabilization of the enzyme of the present invention? 'J's IJe Hibiki 4
To investigate, each substance 1...N1 shown in Table 2 is /X
The enzyme reaction was carried out in a JMU anti-bath solution (pH 7,4).

The results are shown in Table 2.

Table 2 11 E I) i' A: ethyleneno/aminotetraacetic acid 21NENl: N-ethylmaleimide 31 +1 u' kl Is: no-rough LI I
+ -7 Kyuri Henno Eight 4) I) I) '
l”Cnidiethyl/(Pcal/cumate5l-7-u
):4. fi Dehyde 1j Quino 271-hensenonosulfonic acid J-sodium As is clear from Table 2, the enzyme of the present invention is inhibited by about 45% by parachloromercury benzoei I, but not by cupric chloride and diethylnotiosulfonate. It was completely inhibited by carbamay I. On the other hand, the known enzymes are completely inhibited by cupric chloride and diesarditi A carbamate (1, 2), and both enzymes are similar in that they are affected by inhibitors (1). Activation of the invented enzyme nl and ansu i! A chemical agent has not yet been discovered.

(9) Ultraviolet absorption spectrum (see Figure 7!!! 1λ
H1aX ' 2 7' 8 111n
, 3 8 5 11111 , 4 (
i 5 11111 shoulder: 290 nm ImaJKin + 4901u111.1Kin (10) Coenzyme The enzyme of the present invention is heat treated or l 11 is IJI:I acetic acid (
The supernatant obtained by centrifugation after treatment with flavin adenine dinuclease (de(17Δ1)
), and D-amino acid oxidation/der (i's °r・1゛醊Z.

It was found that the coenzyme of the enzyme of the present invention was 1.Δ1).

Also, in thin layer chromatography +<I' 4
Q4 also identified it as FAD.

It was estimated that 2 mol of FAD was present per 1 mol of the enzyme of the present invention.

(11) Polyacrylamide gel electrophoresis and 5
The enzyme of the present invention purified by DS-polyacrylamide gel electrophoresis can be purified by any method.
Reduced i-band.

(12) Molecule] Ministry 1 The molecular weight of the enzyme of the present invention is SENO! 'De Nox G-20
0 (7 Almanoa) Ryoi/manufactured by Chemicals Co., Ltd.) gel il, ji for one person is 135.0 + 10 + - 10,
It was measured as 000.

(13) Isoelectric input 1. When measured by isoelectric focusing using Ampholine (manufactured by +-K B), I) was found to be 6.2.

(141 Crystal structure and elemental analysis of the present invention, <: 1.L L': Crystallization is not completed (1
Measured at 5L It's not L.

(15) Purification method 1 The enzyme of the present invention can be purified by salting-out method, isoelectric precipitation method, precipitation method with organic solvent, adsorption method using diatomaceous earth, live charcoal, etc., various chromatography methods, etc. It can be carried out in combination with the ζ pieces 11 as appropriate. A specific example of the purification method is as shown in Reference Example.

+Bl 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 belong to the genus Streptomyces (31 repformyces) and have the ability to produce the enzyme of the present invention.

Like this 1. A specific example of microorganism A is Tl1, which was isolated from Tonosho-cho, Katori-gun, Chiba Prefecture, and was designated as a single strain.
The tfi Saleta X I + 9-6 strain can be mentioned. The mycological properties of the Jin strain are described below.

A, Microscopic observation Aerial hyphae are linear and have a width of 0.9 to 1. O/I, medium fine 'r) t't small. The sporophyte is twisted by a chain of numerous spores, forming a spiral with 2 to 5 turns.

The spores are slightly oval in shape, with a length of 0.9 to 1. Ox
1, 1 >: 1.2 // Thea', J, Electric t'W
It is observed that the surface has a thorn-like structure. Division of the basal hyphae was observed. Iku (30”C, l 6
The results of macroscopic observation of Ll interculture) are as follows.

(1) Sony Claus/Nitrate Agar Medium Growth is poor.ノ、(Mycelia are grade ? l, ! color and do not invade the agar. Aerial hyphae are powdery and spread thinly (radially) on the agar. The color is gray-brown and forms gray spores. They enter the medium. No pigment formation was observed on 21glucose-asparagine agar medium 11jl The growth was good.], (The hyphae are whitish-yellow and invade the agar and swell slightly.Aerial hyphae are white and poorly grown. , and no production of V and element in the medium was observed.

(3) Growth on glycerin-asparagine agar Jf1 is good.ノ、(The hyphae are whitish-yellow, ′)l; They swell up as they invade the philtrum. Aerial hyphae (J not formed,
Formation of dye into the medium was also observed.

(4) Starch/inorganic salt agar medium No. 41 growth is good. h';t'+'+ The threads are white-yellow and penetrate into the agar. 1. Aerial hyphae (, 1, white, abundant, producing gray spores. 1fζ No pigment formation underground.

(5) Thyronone agar medium Its growth is good. ) A mycelium is white-yellow.

Aerial mycelium is white, abundant, and carries gray spores.

No pigment formation was observed in the medium.

(6) Sakae diameter agar medium The growth is extremely good. The basal hyphae are whitish-yellow and swell as they invade the agar. Aerial hyphae are white and have spores.
No epiphyte is observed. No pigment formation was observed in the medium.

(7) Ys I Wheat, L Agar Medium The cow Y7 is very good. The basal hyphae are whitish-yellow and swell as they invade the agar. Aerial mycelium is white;
It is C9 rich and bears gray spores.

No pigment formation was observed in the medium.

(8) Growth on oatmeal agar medium 1" is extremely good. J, +7 hyphae are white, and they either form fJ in the agar or do not grow on J8 -1.

Aerial mycelia are white and dense, and bear gray spores.

No trace of dye was observed in the medium.

The physiological growth temperature range is 8 to 40°C, with optimum growth around 35°C.

On both tyrosine agar and peptone yeast iron agar, melanin-like pigments are produced, slightly liquefying gelatin and hydrolyzing starch.

D. Assimilation of various carbon sources Various charcoal on Pridham-Godelive cold medium
□Usability of elementary sources is shown in Table 8.

Table 3 So'+ (used) - (not used f11) 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 the medium, the color is whitish-yellow or grayish-brown, and the aerial mycelia are white to grayish-brown, with no production of soluble pigments or melano-like pigments. In addition, starch has strong ice-melting properties.

Based on these results and the assimilability of carbon sources shown in Table 3, Bergey's Manual of Determinative Bacteriology was used.
of Determinative Bacteri
When this strain was identified according to the classification system in the 8th edition (t 974 <+), it was found that this strain belongs to the genus Streptomyces, or that no known species that fully matches the characteristics of this strain could be found. , identified this bacterial strain as a new strain, Streptomyces sp.
1 g-6(3treptomyce, s sp,
X-119-6).

Regarding this strain, we submitted an application for deposit to the Institute of Microbial Technology, Agency of Industrial Science and Technology, in accordance with Notification No. 178 of the Ministry of International Trade and Industry of 1981, and the deposit was made on June 5, 1980, and the accession number was Kenbiyori No. 6560 (FERM P
-6560) is given.

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 mutation induction treatments, such as physical treatments such as ultraviolet rays, Any of the highly productive mutant strains of the enzyme of the present invention obtained by mutating it by the treatment method of (lf) can be suitably used. The purpose of this invention is to utilize the enzyme protein synthesis function of the gene deoxyribonucleic acid (DNA), which carries the genetic information for producing the enzyme of the present invention.
Incorporating such genetic DNA into an appropriate vector,
Transfer to microorganisms of genera other than those mentioned above by transformation, or
Alternatively, the scope of the present invention also includes a method for producing the enzyme of the present invention using a microorganism obtained by genetic engineering techniques, such as incorporating genetic DNA into a colic microorganism by cell fusion using the frodoplast method.

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; is suitable.

The solid medium used for solid culture is no different from that normally used. Zunawaji, what is a solid medium?
, rice bran, corn, rapeseed meal, wheat, rice, rice, etc., or a combination of two or more, and if necessary, the microorganisms used in the present invention are assimilated. Possible nutritional sources, such as glucose, niucrose, 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 sodium salts, potassium salts.

Carunium salt, manganese salt, magnesium salt, %.

Salts such as lead salts, iron salts, phosphates, sulfates, thiamine,
Riboflavin, nicotinoic acid, hunt f7 acid.

Biotin, p-aminobenzoic acid, hitamine IJ12 Z
'j medium supplemented with appropriate growth elements, or these 31
It is a culture medium with various combinations, sizes, and shapes. Such a solid medium may be sterilized or denatured using the infant method, and then a seed culture may be inoculated to perform solid culture. ” - Cultivation methods other than those mentioned above, such as Sufu 1! The liquid culture medium was absorbed into a suitable carrier of funo'-II limb) υ17 (111 Kaisho 49-
No. 14679 (1Q), Even if the method involves inoculating iΦ bacteria and culturing 1, the microorganisms used are still limited;: '/
6+1 which produces l'°i and the enzyme of the present invention well! Can be hired.

The culture conditions ('l) are the optimal conditions for the production of enzymes depending on the type of microorganism used.The deviation is 1: (, not particularly limited. Usually, for example, 20~30'C11ull 5~
7 and culture for 5 to 15 hours.

Collection of Enzyme of the Invention Use The enzyme of the present invention produced by culturing microorganisms can be extracted and separated from the culture, that is, the medium and/or the cultured cells, by an appropriate extraction method and used as is as a crude enzyme ill, or As described above, 1 iL-:+ is purified according to the conventional enzyme purification method to a purification step according to the purpose of use.

The extraction method is particularly limited and is carried out according to 1-116 method 1<. For example, extraction of solid culture is usually done with water or Hi+fill? I will be attacked. In addition, the enzyme of the present invention in the bacterial cells can be extracted by disrupting the bacterial cells using a regular sparge, solubilizing the cells, and extracting the enzymes.

2) Analytical sample In the present invention, the term ``analytical sample 1'' refers to sample f1 which contains or is expected to contain L-glutamic acid as a component thereof, and which has an L-glutamic acid content or its (j i !! (If there is a sample to be analyzed or a reaction system that liberates or is expected to liberate t9L-glutamic acid, and by measuring the change in the 17 glutamic acid content of the system, 21 means an assay for analyzing the enzymatic activity of the system, the content of substances converted into I,-glutamic acid, or the absence of these substances in the system.

Samples classified into the above categories include foods (e.g., soy sauce, amino acid seasonings, various extracts, liquid seasonings such as liquid stock, alcohol-containing foods such as sake, mirin t-ryo, seafood paste products, no sage, ham) Samples classified as q31 include extracts of solid foods (e.g., extracts of solid foods such as
A system containing an enzyme whose reaction product is glutamic acid and its substrate, or a system containing the above system and one or more other Jl or enzyme systems Can be mentioned.

3)1. - Analysis of glutamic acid The method for analyzing glutamic acid according to the present invention involves an enzyme reaction system using the enzyme of the present invention for 1-glutamic acid in a sample, particularly 1-glutamic acid in a sample in which a large amount of aspartic acid coexists. - For glutamic acid, it consists of an enzymatic reaction system in I) H5-6 and a Komura substance detection system that performs constant or qualitative detection of indicator substances consumed or produced along with this.

Enzyme reaction Analysis of L-glutamic acid (Enzyme reaction conditions in i are as follows.

That is, reaction p11 is a reaction in which the enzyme of the present invention is inactivated.
- It is sufficient to use p i-1 that acts sufficiently on glutamic acid, and if the detection system for the indicator substance is dependent on pH, it is preferable to set the pH taking such conditions into account. Generally, the enzyme reaction is carried out in the pH range of 5 to 9. In addition, when the sample t' contains a large amount of di-aspartic acid that substantially affects the analysis of mono-glutamic acid according to the present invention, the enzyme of the present invention can be used to obtain the optimum p I-1 (:I Recently, it has some effect on I--aspartic acid, 1.--glutamic acid for about 1 minute to detect the indicator substance, and ■,--aspartic acid with 'lI. Monoglutamic acid can be specifically separated for 1 hour in such a sample by selecting conditions under which the reaction does not occur.The reaction conditions of pL+ 5 to 6 are suitable as such conditions.

In addition, it is preferable to use various buffer solutions as a reaction medium in order to maintain the pII in the enzyme reaction within a suitable range. 19I does not harm the invented enzyme,
It is sufficient that it does not affect the detection system of the indicator substance. Examples include phosphate buffer, Tris-HCl buffer, acetate buffer, citrate buffer, and heronal buffer.

The reaction temperature is not particularly limited. The optimum temperature for the enzyme of the present invention,
A person skilled in the art can easily determine this by taking into consideration the stability temperature and the detection system of the indicator substance used.

In addition, during the reaction, the enzyme of the present invention can be used with a soluble enzyme, or with a general method such as an entrapping method, an adsorption method, a cross-linking method, or a covalent method.
It is used as an immobilized enzyme that is immobilized by a method.

The mode of immobilization is not particularly limited ([-immobilized enzyme",
Partially edited by Chibata, March 20, 1975, ■Kodansha 1”
,reference). For example, materials for immobilization carriers include cellulose (cellulose), acetyl cellulose, nitrocellulose (collodion), carboxymethyl cellulose, cellulose derivatives, starch, polysaccharides such as dextran, agarose, and chitosan. , proteins such as collagen, fibroin, keratin, albumin, polyacrylamide, polyvinylpyrrolidone, polyvinyl alcohol, carrageenan, alginic acid, xanthan gum, and adult products j'-f! l gel, polyvinyl chloride,
Synthesis of polystyrene, polyethylene, polypropylene, polyurethane, polycarbonate, nylon, Teflon, adsorption resins, ion exchange resins, etc. Inorganic polymers such as polymers, glass, phosphoric acid, ceramics, alumina, etc. are used, film-like, gel shape, granule, powder, microphone UJ
J) Carriers in the form of packets, tubes or containers are used. Examples of immobilization methods include entrapment of the enzyme of the present invention with trivalent Kel-Z, adsorption of porous or ion-exchanged substances, covalent use on carriers by peptide method, alkylation method, diazotization method, etc. bond, glutaraldehyde,
An appropriate method can be employed such as crosslinking the polyfunctional 11 of hexamethylene diisonane-1.t5 to a carrier using a crosslinking agent or crosslinking 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 tightly attached to the electrode as a membrane-like immobilized enzyme, or it is attached to the electrode surface. When the enzyme is directly immobilized and the reaction is carried out in one reactor type, a column type reactor with a granular immobilized enzyme column, a tube type reactor with the enzyme immobilized on the inner surface of the tube, or an enzyme immobilized on the inner wall of the measurement vessel is used. can be used.

Detection of Indicator Substances The substances used to analyze 17-glutamic acid by the present invention are oxygen consumed and produced by an enzymatic reaction, as well as hydrogen peroxide, ammonia, and α-ketoglutaric acid.

Detection of each indicator substance is carried out by an arbitrary method, and the present invention is not limited to that detection method.In other words, the detection power is generally determined by a known method, but it should be further developed in the future. It is considered that various methods can also be adopted.

゛ In addition, detection system 1 for 1 small indicator substance is as follows: L-glutamic acid is analyzed qualitatively and quantitatively (1) based on the detection process of each indicator substance and the detection results (1) Detection Methods for detecting consumed oxygen include Warburg Kengetsu, Method (Biochemistry Experiment +!
pp. 5-41, ■Tokyo Kagaku Doujin, 1975 (August 201
7I published) and the oxygen electrode method (Oxygen measurement by electrode method, edited by Bunji Hagiwara, Kodan Jl:, 1977 (lul January 201), Mitsuyuki), and in the present invention, either method is adopted. Good too.

The electrodes used in the oxygen electrode method include platinum, gold, iridium, and Q]1+i as working electrodes, silver, silver/silver chloride thread, and saturated JJ IJ solution as the test solution, Teflon membrane, and polypropylene as column electrodes. A composite electrode that is integrated with an oxygen-permeable electrode membrane such as a membrane or polyethylene membrane (diaphragm oxygen electrode such as a crack-type electrode)
Alternatively, a separate electrode may be used in which the working electrode and the reference electrode are connected by a salt bridge. The method can be a polarographic method or a galvanic cell method.

When measuring oxygen with an oxygen electrode, there is no measurement deviation due to the power of 7+H passing through the dissolved oxygen of the enzyme reaction solution. In addition, an enzyme electrode consisting of an oxygen electrode and a wood-invented enzyme mounted on the electrode surface, such as an immobilized enzyme or
'A soluble enzyme covered with a transparent membrane may be measured using an enzyme electrode attached closely to the electrode membrane of a Clark type electrode.

(2) Detection of hydrogen peroxide Electrochemical analysis, spectroscopic analysis, fluorescence analysis, and chemiluminescence analysis are known methods for detecting hydrogen peroxide produced. Either method may be adopted in the invention.

For electrochemical analysis, a hydrogen peroxide sensing electrode with the same structure as the oxygen electrode described above was used, and the current method was used.
The most common method is to measure 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, peroxider-V or molybdic acid 1 can be used together with the enzyme of the present invention.
A method can also be used in which hydrogen peroxide and iodine ions are reacted using a hydrogen peroxide decomposition catalyst such as No. 1.11, and the decrease in the activity of iodine ions on the electrode surface is measured using an iodine ion electrode. Incidentally, the above method is suitably used as an enzyme electrode in which an immobilized enzyme having the enzyme of the present invention and peroxidase or a catalyst is attached to an iodine ion electrode.

Spectroscopic analysis methods include: (1) peroxidase method, in which an oxidizable coloring agent is reacted with hydrogen peroxide in the presence of peroxidase or a substance showing similar activity, and the absorbance of the produced melon is measured; (2) alcohol The aldehyde is reacted with hydrogen peroxide in the presence of catalase, the resulting aldehyde is introduced into a coloring system, and the absorbance of /I: rΔ Immune is measured, or the aldehyde dehydrogenase is reacted with nicotinamide adenine dinucleotide I' ( A method that measures the production of reduced NAD (NAI) I+) in the presence of NAD).
Ze method, and (3)' self-flVl/xylenol orange system, Ti(IVI/4-(2-pyridylazo)
There are chemical methods using resorcinol, V m /kircinol orange, etc.

In the peroxidase method of (2), the 9-color reaction 1; 0-tolidine, 0-toluidine, (]-aminophenol, 2.4-dichloroindophenol, benzidine, 8.8: 5.5'-ftraalkylhenzidine (8,3: 5.5'-tetra methylhenzidine, etc.), 4-methoxy-1-naphthol,
2.2'-amino-di(3-ethylbenzthiazoline-6-sulfonic acid), guaiac butter (guaiafur)
, N-(4-antipyryl)-aniline derivative IN-(
(4'-antipyryl)-2-carboxine-4-hydroxyaniline, etc.), p-hydroxy7phenylacetic acid WN
, N-dimethyl-p-phenylenediamine (7'l).

In the latter method, 4-aminoantipyrine, a 4-aminoantipyrine derivative with 4-aminoantipyrine Amitona S, 4-aminophenazine, 4-amino-N,N-dimethylaniline, p-phenylenediamine, 4-Amino-N.

N-9 ethyl-m-toluidine] phenylenediamine derivative, 3-methyl-2-hen'notiaso I-hydrazone (used as MBTl, etc.)
In particular, phenol, notticol, renowalcin, hydroquinone, cresol, fi-col, pyrogallol, orno/l)/7 L) uphenol, I)-promophenol, 2.4-nochlorophenol, 2.4-Sifromophenol, 2゜4,6
- Tribromophenol, aniline', N,N-dimethylaniline, N,N-diethylaniline, N-ethyl-N-(3-methyl-lephenyl)-M acetylenediamine, 3-acetamino-N, N-diethyl Aniline,
N,N-ethyl-111-1・'Luidine,N-ethyl-N-(2-hydroxy73-sulfopropyl-m-
Toluinone, N-RIJsilyl N-ethyl-m-toluinone, p2-methylaminophenol, 0-aminophenol, 111-aminophenol, p-methylaminophenol, 2-chloro-6-methylphenol lolo-3 -Methylphenol, 3.5
+. Phenolic, aniline or toluidine compounds such as 12-hydrogyabenzenesulfonic acid, or 4-halogeno-1-su71--2-sulfone a
(4-chloro-1-1-phtho-2-sulfonic acid), 1-naphthol-2-sulfonic acid, 2-amino-5
-Naphthol-7-sulfonic acid, 2,4-Ivy U-Rho 1
-naphthol, 1-hydroquino-2-naphthoic acid,
4.5-dihydroxynaphthalene Naphthol compounds such as 2,7-disulfonic acid, naphthylamine or its derivatives, quinoline compounds such as hydroxyquinoline and aminoquinoline, etc. can be used as color formers. Examples of suitable combinations of 4-aminoantipyrine and phenol, N,N-dimethylaniline, N,N-diethylaniline or N-diethyl-
combination with ml luidine, or 3-methyl-2-hensothiasolinohydrazone and N
, N in combination with dimethylaniline. The present invention is not limited to the types of these color formers or color formers and couplers, but oxidation can also be used quantitatively.

Further, as peroquinodase, an enzyme derived from horseradish (horseradish) or sweet potato is usually used, but any enzyme that exhibits peroquinodase-like activity may be used, and is not particularly limited. Furthermore, it may be a catalyst that exhibits the same t41 activity as peroxidase.

In the catalase method described in (2) using a coloring system, methanol is usually used as the alcohol, and hydrazone (for example, 3-methyl-2- Henzothiazolinone hydrazone, 4-amino-3-hydrazino-5-mercapto = 1.2.4 ~ Triazole (method of reacting with AI (MTl etc.) to develop color, reaction of formaldehyde with acetylacetone and ammonium salt) Another method is to use curtathione and guide it to oxidized tartathione using hydrogen peroxide-glutathione peroxidase/dase, which is then converted to oxidized tartathione using a method (Analytical Biochemistry (4nal)).

Biochetn, ) 76, 184-1
91 + 1 976 )), there is also a method of oxidatively decolorizing .fndigocarmine with a copper ion-histamine system and determining the color intensity from the decrease in chromaticity.

As a fluorescence analysis method, 1: Movanillic acid is reacted with hydrogen peroxide in the presence of peroxidase to produce fluorescent 2,2'-dihydroxy-3,3'-dimethoxybiphenyl-5,5' - Conversion to noacetic acid and measurement of fluorescence intensity can be mentioned. In a similar manner, p-hydroxyphenylacetic acid, diacetylfluorescin derivatives (cyacetylfluorescone, diacetyldichlorofluorescone I Yoto 1, etc.) may be used as the luminescent reagent instead of homovanillic acid. , scopoletin, and 3,5-diacetyl-1,2-dihydroxy 7) are oxidized to non-fluorescent substances by hydrogen peroxide/peroxidase/dase, and the decrease in fluorescence is measured. There is a way to do that.

For chemiluminescence analysis, luminol is used as peroxinotator.
A method of measuring luminescent nails by reacting with hydrogen peroxide in the presence of enzyme (JP-A-5771399, JP-A-57-
71400). In a similar manner, instead of luminol, inluminol, pyrogallol, bis(2,4
, 6-1-lichlorophenyl) oxalate may be used, and instead of peroxy/dase, hemoglobin,
Hematin, hemin, iron cyanide (potassium, cobalt chloride, etc.) may be used as catalysts.

(3) Detection of ammonia Ammonia can be analyzed by methods such as the microkeltal method, the Nessler method, the indophenol method, the ninhydrin method, and the phenosaffron method. In addition, electrochemical analysis methods include analyzing ammonium ions using a cation-selective electrode, or analyzing ammonia gas using an ammonia gas electrode consisting of a glass electrode equipped with a hydrophobic sludge-permeable membrane. Good too. 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 7-ketoglutaric acid + 1,8-methyl-2-benzothiazo, tp, hydrazone (M I-3T I-1)
2.4- Method of measuring the absorbance of the product by reacting with
A method of reacting with dinitrophenylhydrazine and measuring the absorbance of the product, a method of reacting with 0-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, 1: I, its form is not limited,
It may be a soluble ↑/1 enzyme in solution, frozen, powder or granule form, and as mentioned above, various h°
The enzyme may be an immobilized enzyme immobilized on a film-like, gel-like, granular, powder-like, microcapsule-like, tube-like, or container-like carrier by a method. In addition to the enzyme of the present invention, liquid or powdered acid buffers, Tris-hydrochloric acid buffers, vinegar/acid buffers, citric acid buffers, buffers with Bess buffers f and g, and salts. (sodium chloride), sugar mfi (
Sucrose sud), polyhydric alcohols (glycerol, propylene glycol, sorbitol), coenzymes (FAD, etc.), other appropriate stabilizers, surfactants, etc. may be added.

When analyzing L-glutamic acid, the reagents for analysis are as follows:
Depending on the various detection methods described above, they are used so as to obtain the necessary enzyme'/+1 characteristics. 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 consists of an analytical reagent containing the enzyme of the present invention as 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 reagents include, for example, a combination of a compound and a coloring agent or a coloring agent or the coloring agent Onibino J. or a combination of a catalase-like active substance and a reagent necessary for an alcoholic color system or a reagent necessary for a conjugated enzyme system, a combination of a var-oxidase or nomer-ockindase-like active substance and a fluorophore, /<- Examples include combinations of peroxidase or peroxidase-like active substances and luminescent reagents. Specific examples of these reagents are clear from the description in +iif (-Detection of hydrogen peroxide 1).

Furthermore, when using ammonium or rt-ketoglyric acid as the indicator substance, the necessary detection reagents can be similarly combined with the analytical reagent containing the enzyme of the present invention to form an analytical kit ( , “C-kiru,” In addition, it contains the enzyme of the present invention.
The analytical reagents to be used and the detection reagents described above are mixed together and
It may also be used as a reagent for
(1: In the case of 1), each component may be divided into appropriate combinations (1).Also, these may be prepared in the form of a melt or as a powder reagent (4). It may be contained in a suitable support such as paper or film, and prepared as a test paper or a 1-minute IJ+ film).

In addition, the analysis kit of the present invention is 1 piece! -, l note o'r
ill <'H + In addition to the i reagent, a standard reagent containing glutamic acid - and constant I+ may be attached.

Analysis kit of the present invention! An example of a suitable example is a kit for analyzing 1,-glutamic acid (by spectroscopic detection of hydrogen peroxide).
, 02 units <U) or more than 1.7/tes), Nog oxidase 1-10 tJ/test, color former, 1,4-aminoantipyrine and phenol or It N.

When N-dimethylaniline is used, the amount of these reagents is 1 mole or more, preferably 2 moles or more, based on the amount of hydrogen peroxide produced.

4) Specific Examples Below, concrete examples of the analytical method, analytical reagent, and analytical kit of the present invention will be shown as examples. 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 (Preparation test IA of 11 kits: L-glutamate oxidase 1 mg (
] OU/'Wt), Bar No. 4 - Sodase 5m
1 (lo.

U/■ ; derived from Nishi IY wasabi, manufactured by Toyobo ■) and 4
- 0.2 M potassium phosphate buffer (+) H so that 40tRg of aminoantipyrine is contained in one reagent bottle.
, 6, 5), and lyophilized using a conventional method.

Reagent B: ■ Dissolve 40 mg of phenol in o.
Dissolved in IM potassium phosphate buffer (+)H6,5),
Placed in a reagent bottle as IOOml.

(2) Procedure: Put L-glutamic acid in a test tube.
Dispense 0.1 ml of each of lk and water, and dissolve the lyophilized product in 100 ml of either 1 or 2 of reagent B into each of the above test tubes. Add 0.9 d and shake aerobically at 37°C.1. Turtle is 1゛.

1. Incubate for 20 minutes.
When using Ichiki ■ as j, 500 nm,''
When using *, absorbance at 565'nm is 11! I guessed>jI. These test m lines are shown in Figure 8 (reagent 13 force (1
・J, ll.

) and Figure 9 (for reagent B or (2)).
.

Example 2 (1) Preparation of reagents Coloring reagents: Phenol 20v, 4-aminoantipyrine 20+17 and horseradish baroquinoda-W8t
50 ml (7
) 0.2 M potassium phosphate buffer (1) H7,4)
dissolved in.

L-glutamic acid oxidase SEI purified enzyme 300 t
t'J (55[J/s+g) to 80 wtt (1
) 0.02 M I) Potassium phosphate buffer (+) H7
, 4) (0.050/shoulder t).

(2) Step 6: Dispense 0.8 ml of the coloring reagent into test tubes, add 0.1 ml of L-glutamic acid ox/dase solution, and incubate at 37°C.
Place in a constant temperature bath and heat for 5 minutes (7). +4+ '7L
- Sodium glutamate solution (0-2 //lTl0
I7*t) or sample name T#t (a solution prepared by diluting various soy sauce products 150 times with water) and adding 0.1*t to j:<
Mix and incubate for 20 minutes with aerobic shaking at 37°C.

The absorbance at 500 nm was measured using a blind test as a control.
A calibration curve shown in Figure 0 was created. The quantitative value of monoglutamic 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 ferrule phthalein using Technicon Auto Analyzer). A comparison with the L glutamic acid constant resistance value in Table 4 (small after Example 3) is shown in Table 1.

Note that the correlation coefficient γ is 0.997. Regression formula y 1.00
It was 5x-0,575.

Example 3 0.1 ml and L of 0.1 M potassium phosphate buffer (pH liver derived, manufactured by Sigma Chemical?i) solution in a test tube
-Glutamate oxidase (0.6U, / Shoulder υ solution 0
．． Take 1 ml and add it at 37°C for 5 minutes! ! did
To this was added standard L-glutamate solution (0 to 5
71 mol/*t) or 0.1 we of a sample solution (same soy sauce dilution solution as in Example 2) was added to start the reaction. After shaking aerobically for 37°Cl2O minutes and incubating from 1↓, 2596 tric (10 acetic acid 0
．． The reaction was stopped by adding 1 ml. I in the reaction stop solution
Mlli acid buffer (1) H5,0) 1.9 shoulder t and 01% 3 Methyl-2-benzothiazo 1ψnohydrazone hydrochloride solution 1 (kO, 8 ml) was added, and after stirring, ino4 was heated at 50°C for 30 minutes. After cooling to room temperature, the absorbance at 816 nm was measured using a blind sample as a control, and a small sample 111 line was created in Figure 11. From this sample 111 line, t was determined.
The constant value of glutamic acid in two samples was 1. A comparison was made with the quantitative value of rutamic acid in the sample solution according to the conventional method using glutamic acid decarboxylase [Table 4 shows the results]. Note that the correlation coefficient γ-0,! J 96 , regression equation y=1.0
2 G

Table 4 Example 4 (1) Preparation of reagents [Q Test a: Phenol 80 mv, 4-aminoantipyrine 30 mg, horseradish peroxidase 4
mg (100Uy'ml) and L-glutamate oxidase 1 mg (10u/ig) at 0.2
M Norium phosphate buffer (1) H6,5) 50we
dissolved in.

Substrate solution A: L-aspartate-+-thorium 20
0 mfl and 15 rng of α-ketoglutarate oI
M potassium phosphate buffer solution (1) l-1'1.0>
It was dissolved in 10 mr.

Substrate solution B: L-Alania 140 m (l and α
-151 ffg of ketoglutaric acid was dissolved in 10 ml of 0.1 M potassium phosphate buffer (pH'7.0).

(2) Operation method ■ Activity measurement of glutamic acid/oxa TJ Q' + acid tranosaminase (GOT) Transfer 2 ml of substrate solution A into a test tube and add ゎtsu semi-enzyme solution (manufactured by Boehringa - Yamanouchi, 380 U/ mg) 0
, 1 tnt and incubation at 376C for 80 min.
:I-to-IL, then 25% trichloroacetic acid 0.1
ml was added to stop the reaction. IM potassium phosphate buffer (pH 65), 0.5 at of a coloring reagent containing IMt and L glutamate oxidase were added to the reaction termination solution, and the mixture was incubated at 37°C for 20 minutes. The absorbance at 5001 m was measured using Aken as a control, and a test II) line (not shown in FIG. 12) was created.

■Glutamic acid/pyruvate trans τ minase (G
P ``]'') Activity Measurement Test 9'1 Take 2 ml of the quality solution BO, and add the standard enzyme solution (Herrin made by Yamanouchi ■, 1.40 U/ig).
After adding 0.1 ml of the mixture and incubating at 37°C for 30 minutes, the reaction was stopped by adding 0.1 ml of 25% Trilit IJ acetic acid. Reaction stop l (I in k
, - Glutamic acid content m was measured in the same manner as in ■, and the calibration curve shown in Figure 13 was created.Example 5 L-glutamate oxidase (0.5 U/shoulder t)
．． b O, I M Re/u acid power') Ram 1m t
! Iif T (1, (pH 5, 5) 1 ml at 30°C
A 50 IIe 1. Oxygen consumption was measured using glutamic acid standard J+ solution (0-21 tmol/H1), and the line shown in Figure 14 was drawn.

Example 6 0.5 ml of a 25 U/mO solution of L-glutamic acid oxy/tase (25 U/mO) was applied to a porous nitrocellulose membrane (TOIY;
+1 Gakusangyo ■, TM-5i pore size Q, l/127
/, diameter 25 order, film thickness 140 ptn)
...The suction 9 was fixed by suction to obtain an L-curtamic acid oxide/dase membrane preparation.

After cutting the above immobilized enzyme membrane into a circle (50 mm in diameter),
It was attached to the gas permeable membrane (Teflon membrane, membrane thickness IQ 1i111) of a diaphragm oxygen electrode (required by Ishikawa Seisakusho, L12 type), and a cellulose dialysis membrane was attached to the immobilized enzyme membrane. was manufactured.

Using the same sensor, a known a degree (005 to 1,011 m0
'7Me) +7) L -g /l
/ 9 Mi 7 Acid Boo L'
The current reduction value for the J'/"a"+" solution was measured, and a test line shown in No. 151x1 was created.

Example 7 A 0.1 mt solution of L-glutamic acid (4481 J/rrv) was dropped onto a 801 nm x 3 Q my square film (film thickness: 30 μm) and dried, and 25% glutaraldehyde was added to this. 0.1 ml of solution
was infiltrated and dried overnight at 4°C to obtain an L-glutamate oxidase-immobilized se117-non membrane.

The immobilized enzyme membrane described above was attached to the diaphragm and the elementary electrode I in the same manner as in Example 6. When the current reduction value with respect to the L-curtamic acid standard lrk was measured in the same manner as in 6, a linear relationship was obtained in the concentration range of 005 to 1.

The response to sodium L-glutamate solution (0,811M) at each pH was measured (30°C) using the sensor described above, and the results are shown in Figure 16.

As is clear from FIG. 16, the optimum pH of the sensor was close to pH 6.5 to 9.5.

In addition, the responsiveness of the above sensor to various amino acids was measured, and the results are shown in Table 5. The concentration of each amino acid solution was 1.0 mM, and the reaction was carried out at pH 7.0 (fl,
] M phosphate buffer solution) was used.

Table 5: The above sensor was stored in 0.02M phosphate buffer at 3°C, and its responsiveness to 1 cL-sodium glutamate solution was measured in the same manner as above in each case. The original responsiveness was maintained.

Once tested, the immobilized enzyme membrane is stable at 3°C for at least one month.

Reference Example: Put 20 liters of bran and 16*/ of water into a 500-liter Erlenmeyer flask and sterilize under pressure at 120°C for 30 minutes.
Sp.
iHlte1ili +? j I prepared it.

25 51 volume Erlenmeyer flasks 200 bran each
LJ and water 160*f to 5.11.120°C13
After autoclaving for 0 minutes, the above-mentioned inoculum was inoculated, and after culturing at 28°C for 211 minutes,
The cells were further cultured for 2 weeks at 1.1 m.c.

The resulting culture was soaked in 37.5 μl of water for 1 hour, filtered, and further passed through a diaphragm.
Approximately 34e of I'11 enzyme solution was obtained. Add monium sulfate to this Ill yeast juice until saturation is 5096, collect the resulting precipitate by centrifugation, and add 0.02M acetic acid solution to Ilf (Ill).
'll 5.5) Dissolved in 3.91 °C and heated to 3.91 °C at 57°C.
After heating the enzyme solution for 20 minutes and cooling it to 5°C or more, add 2x 111 of pre-chilled ethanol, collect the precipitate by centrifugation, and collect the precipitate at 0.02 M. Re/
uM6 [jiJ1 solution (+) H7,4) dissolved in 2e (2,
The same buffer τ- was dialyzed overnight. The precipitate generated during dialysis was removed by centrifugation, and the soil-seine solution was diluted with the same buffer 111. Te・I'?
Ir-modified I) EAE (di-1-thylaminoedyl-cellulose column (8, Fix 50z) was passed through, and the absorbed enzyme was eluted using the same buffer containing 0.85 M of sodium chloride. The active fraction was collected and added to 0.05λ4 acetate buffer (+)H5,5) containing 0.05M NaCl.
I underwent dialysis. This dialysate solution was re-formulated with the same buffer solution and treated with an E-A E-Sepharose CL-6B (7Al, No. Phi,/Gemicalson 1) column (1).
2・l0nn) and inhale? 'F L'enzyme salt 0,
05-fl, 75 M tree: j”,'/”
, i -> 17 was eluted. Eluted active fraction≦・fl4th, after dialysis concentration, Sephadex G-200
Made by F No. γ Luminaire Finechemirils 11) l/J
Perform 11 filtration using 2.5x 120z filter, collect the active fraction, and after concentration, reduce to 0. fl 2 N
+ Phosphoric acid power 11 1. 1 in buffer (pfl 7.4)
, dented l. This dialyzed fluid was centrifuged, and the supernatant was passed through a precision sieve and freeze-dried.1. - Purified specimen of glutamate oxidase (specific activity f'l: 55.] U7/my protein 1 yield 18.4%) 80 ■ was obtained 1.

[Brief explanation of drawings]

FIG. 1 shows the action pH axis range of the enzyme of the present invention (solid line) and the known enzyme (dotted line). FIG. 2 shows the H4iij range (held at 37° C. for 160 minutes) of the enzyme of the present invention (solid line) and the known enzyme (dotted line). Figure 3 shows the stable pH range (maintained at 45°C for 115 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 stable temperature ranges of the enzyme of the present invention (solid line) and the known enzyme (dotted line, ○-○). In FIG. 6, Moum has a pH of 5.5, and -O has a pH of 7.5.閤-哩 is pH
The degree stability curve is shown by ^ under each condition of 9 and 5. FIG. 7 shows the ultraviolet absorption spectrum of the enzyme of the present invention. Figures 8 and 9 show the kit of Example 1, with phenol solution or dimethylaniline solution as reagent B.
The graphs show the test lines of mono-glutamic acid using Ik. FIG. 10 shows a calibration curve for glutamic acid in Example 2. FIG. 11 shows a calibration curve for monoglutamic acid in Example 8. Figure 12 shows the test 1i of GOi' activity +'l in Example 4.
The j line is shown. FIG. 13 shows G P i'/+l'l in Example 4.
(I11 line of 'I' is shown. Figure 14 shows the calibration curve of Shichirutamino acid in Example 5. Figure 15 shows the calibration curve of Shichirutamino acid obtained using the enzyme electrode of Example 6. The acid calibration curve is /j<. Figure 16 shows the responsiveness at each pIT of the enzyme electrode of Example 7. 1 figure 2 figure 5 figure 6 figure warm
o ttH Procedural amendment (voluntary) August 23, 1980 [1 1, Indication of case Patent application No. 11 filed August 210, 1981 2, Title of the invention (old) L - Analytical method and analytical reagent for glutamic acid and Analysis Kit 3, Relationship to the stand case that is being amended Patent applicant address (zip code 288) 2-10-14 Shinsei-cho, Choshi-shi, Chiba Prefecture Column for the title of the invention in the application to be amended and description Revision 5 of the invention Contents of the amendment 2) Specification, page 25, 4-711 (this 1' +111
Polyacrylamide gel electrophoresis and 5DS-polyacrylamide gel electrophoresis The purified enzyme of the present invention showed a single band in both methods. The enzyme of the present invention purified by polyacrylamide gel electrophoresis showed a single product. ” he corrected. 3) 1'-0,05U/Shoulder/in the first line of page 57 of the specification
Correct 1 as r O, 55U/*t, I and Jj. Procedural amendment I (spontaneous) September 27, 1980 Kazuo Wakasugi, Commissioner of the Patent Office1, Indication of the case Patent Application No. 145346 of 19822, Name of the invention: Method for analyzing L-glutamic acid, analytical reagent, and Analysis kit 8, relationship with the amended person case Patent applicant address (zip code 288) 2-10 Shinseicho, Choshi City, Chiba Prefecture Telephone number 047
9 f22) 0095 (representative number 4, in the specification subject to amendment) column for detailed explanation of the invention and column for brief explanation of drawings, drawing 5, content of amendment 1) specification il + page 6, line 13 [1 ni rL-algine”
Correct the statement as “L-arginine J2” Details;
! ) On page 7, line 8, 1K-1 is corrected to ``equilibrium 1.'' 3) On page 17 of the specification, lines 6-5 from the bottom, 1-L-glutamic acid. .] is I■, -glutamine 1 and i
1 Correct. 4) On page 24, line 4 of the specification, the phrase ``For 1 carbamate'' was changed to ``For carbamate'' by 35 degrees. 5) In the opening of the 6th line on page 40 of the specification, change the text ``1 type'' to ``1 type''. 6) On page 58 of the specification, lines 17-18 and line 19, 1-sample solution ``1-There are many things in 1-sample'' and 8I
Correct. 7) In Table 4 on page 59 of the specification, the text "1-sample solution" is corrected to "1-sample". 8) In specification box page 61, line 14 i'+II, correct "potassium phosphate" to "acetic acid" 9) Description of 1 Example 5 up to page 61, line 19, 1 of the specification Following this, add the following text. [The quantitative value of L-glutamic acid in the sample (various soy sauce products) obtained from this calibration curve was calculated using the conventional method using L-glutamic acid decarboxylase ((l)
A comparison with l'f is shown in Table 5. Note that the correlation coefficient γ-0
．． 958. Regression formula Y == 0.980X+ 6.11
Met. Table 5 10) Table 1155 in page 63, line 16 [] of the specification is corrected to 1-Table 6-1. 11) In Specification 71, page 63, line 16 b to last line, ``In addition, each amino acid solution ... line -] -1'' is replaced with 1. 2.01
A solution with a concentration of 11M was used, and L-glutamic acid was 0,4
Measurements were made using a solution with an rnM concentration, and the measured values were converted to calculate relative response values. The reaction is pi-15,5(0,
'IM acetic acid-silium acetate buffer]. ”
12] Table 1-5 on page 64 of the specification is deleted, and Table 6 of 1-1 is added. Table 6 13) The following sentence is added as "Example 8" after "1 Example 7" on page 65, line 5, up to 11 of the specification. [Example 8 ■, -0.1 M acetate buffer (p
H5, 5) Seal 1ml into the cuhette of a Clark type oxygen electrode circulating constant temperature water at 30°C, and
A glutaminase marker derived from E. coli! ink (
41 nU to 201 nU) was injected, the oxygen consumption rate was measured, and a calibration curve shown in FIG. 17 was created. Similarly, 50 m of soy sauce koji suspension (soy sauce JifilO'/
l of 0.1M phosphate buffer (p) 47.0)
00 rpm. After homogenizing for 5 minutes, add the same buffer and
l/closed liquid) 50 tt (inject l terV
The A element consumption rate was determined as 7Il11, and the glutaminase activity per gram of soy sauce koji was determined.
Correct 11 to read 1-equilibrium] instead of 1-equilibrium. 15) Specification (brief explanation column of drawings) No. 70, No. 15
The following explanation regarding FIG. 17 is added from line 11 onwards. "Figure 17 shows the calibration curve of glutaminase activity in Example 8." 16) In the graphs of Figures 12 and 13, "μU/tube" was corrected to 1 mU/tube 1. , attach the corrected drawing as a separate sheet. 17) Add a new figure 17 and attach it as a separate sheet.

Claims (1)

[Scope of Claims] 1) It has extremely high substrate specificity for L-glutamic acid in analytical sample 11, does not substantially act on amino acids other than L-glutamic acid, and has a high stability quotient. 1. A method for analyzing monoglutamic acid, which comprises causing glutamate oxidase to act in the presence of oxygen and water, and detecting the consumption of oxygen or the production of hydrogen peroxide, ammonia, or α-ketoglutarate accompanying the reaction. 2) L-glutamic acid quintase has a pH of 5.5°15
The assay method according to claim 1, which is an enzyme that exhibits stability such that the activity is low at 65'C under holding conditions for 10 minutes. 8) Along with L-glutamic acid, L- For monoglutamic acid in the analysis sample containing aspartic acid, we use highly stable L-glutamate oxidase, which has extremely high substrate specificity for L-glutamic acid and substantially acts on amino acids other than L-glutamic acid. When reacting in the presence of oxygen and water, react at pH 5 to 6,
1. A method for analyzing L-glutamic acid, which comprises detecting the consumption of oxygen or the production of hydrogen peroxide, ammonia, or a-ketoglutaric acid during the reaction. 4) L-glutamate oxodase pH 5.5. The analytical method according to claim 3, which is an enzyme having stability such that its viscosity does not decrease up to 65°C under holding conditions for 15 minutes. 5) A reagent for analyzing L-glutamic acid, which contains a highly stable L-glutamate oxidase that has extremely high substrate specificity for L-glutamic acid and acts substantially on amino acids other than L-glutamic acid. 6) L-glutamate oxtase pH 5.5. 65°C or 111°C under holding conditions for 15 minutes
An analytical reagent according to claim 5, which is an enzyme having stability such that t'1 does not decrease. 7) A patent comprising L-glutamate oxidase and a buffer suitable for the reaction of the enzyme. Claim i 8) The analytical reagent according to any one of claims 5 to 7, which is an immobilized enzyme in which L-glutamate oxidase is immobilized. 9) It has extremely high substrate specificity for L-glutamic acid, has virtually no effect on amino acids other than L-glutamic acid, and has a stability of 1°. ;》Kit for analyzing monoglutamic acid 1, consisting of glutamic acid oxidase and a reagent for detecting the reaction by the enzyme. 10) L-glutamate oxidase is p. I-I,
5. 5. Activity is low at 65°C under holding conditions for 15 minutes}! The analytical Kira 1 according to claim 9, which is an enzyme having good stability. 11) Reaction detection reagent or f', which is a reagent for detecting hydrogen peroxide, ammonia, or α-ke1-curcuric acid.
l'N'l The analytical kit according to claim 9 or 10. 12) Claims 9 to 1, wherein the reaction detection reagent is a single color former or a combination reagent of a color former and a coupler.
Minute fJi III Kit I・0 as stated in any of paragraph 1