JPS5934882A - Biosensor - Google Patents

Abstract

PURPOSE:To obtain a biosensor by using L-glutamic acid oxidase having extremely high substrate specificity to L-glutamic acid, essentially inert to the other amino acids, and having high stability, as the receptor for L-glutamic acid. CONSTITUTION:The biosensor for the specific analysis of L-glutamic acid in a specimen is composed of an enzyme acting as a receptor for L-glutamic acid, and a transducer to detect the chemical or physical change in the specimen caused by the action of said enzyme and transmit the change as an electrical signal. The enzyme used in the present biosensor is L-glutamic acid oxidase having extremely high substrate specificity to L-glutamic acid, essentially inert to amino acids other than L-glutamic acid, and having high stability, e.g. L- glutamic acid oxidase produced by Streptomyces sp. X-119-6 (FERM-P No.6560).

Description

Detailed Description of the Invention (1) Background of the Invention Technical Field The present invention relates to a biosensor for specifically analyzing L-glutamic acid in an analysis sample. The present invention also relates to a biosensor characterized in that a novel enzyme L-glutamate oxidase is used as a receptor for L-glutamate.

Prior Art Conventionally, as a biosensor for analyzing L-glutamic acid, (1) glutamate dehydrogenase is
- Enzyme electrode that is used as a glutamate receptor part and a cation electrode as a transducer part (
Anal ChimJ
Acta. ), 56, 333 (1971)) and (
2) Microbial electrode (Anal Chim, Acta, 116, 61) that uses freeze-dried cells of E. coli as a receptor for L-glutamic acid and a carbon dioxide gas electrode as a transducer.
(1980)) is known.

(D enzyme electrode has extremely poor enzyme stability (2 days)
, since a cation electrode is used, sodium ions,
It is not practical because it is easily affected by coexisting cations such as potassium ions. The microbial electrode (■) uses immobilized microbial cells, so it has excellent stability (for 3 weeks,
1,500 times or more), but under aerobic conditions, carbon dioxide gas is produced by the respiration of bacterial cells, which affects the measurement. In order to eliminate such effects, it is necessary to inhibit the respiratory action of the bacterial cells by blowing nitrogen gas, and to remove carbon dioxide from the sample. Furthermore, since this microbial electrode does not have high substrate specificity, it is thought that it can only be used for rough measurements such as fermentation process control.

Furthermore, an enzyme electrode using L-amino acid oxidase as a receptor part is also known (Analytical Chemistry (Anal, Chem), 47).

1359 (1975), etc.). However, the conventional L-amino acid oxidase used in such enzyme electrodes is difficult to act on L-glutamic acid, and even if the enzyme electrode described above is used, it is not possible to specifically analyze L-glutamic acid. Ta.

Recently, L-glutamic acid with high substrate specificity for L-glutamic acid
Amino acid oxidase is produced by Streptomyces (Streptomyces).
ptomyces; hereinafter sometimes abbreviated as "S". )
It has been discovered that it can be produced by culturing a microorganism belonging to the genus, specifically Streptomyces violetscens (see Japanese Patent Application Laid-Open No. 57-43685). 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 set as 100, L
-84 for glutamine, 6 for L-histidine
．． It shows a relative activity of 8 and no substantial activity against other amino acids.

(2) Optimum pH pH 5-6 (3) pH stability Stable in the range of pH 3.5-65 (held at 37°C for 1 hour).

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

(5) Effect of inhibitors Almost completely inhibited by mercury ions, copper ions and diethyldithiocarbamate.

Although the above-mentioned published patent publication does not disclose whether or not the above-mentioned known enzymes can be used in a biosensor for specifically analyzing L-glutamic acid, it is possible to use the known enzymes in the receptor part of a biosensor. Even so, various problems may arise. In other words, although the known enzymes have higher substrate specificity for L-glutamic acid than the previously known L-amino acid oxidases, they still show distinct activities for other amino acids as mentioned above, and the coexistence of these amino acids It cannot be used as a biosensor for specific analysis of L-glutamic acid. Furthermore, known enzymes do not have high stability, and are not necessarily considered to have good storage stability and use stability when used in the receptor part of a biosensor. Furthermore, if copper ions are present in the analytical sample,
Known enzymes are thought to be significantly inhibited, making analysis difficult.

As described above, conventional L-glutamic acid that uses oxidase as a receptor to specifically identify L-glutamic acid
There was no biosensor for glutamic acid analysis, and no L-amino acid oxidase that could satisfactorily achieve this purpose. .

[■] Summary of the Invention The present invention comprises an enzyme as a receptor for a substance to be detected, a transducer that detects a chemical or physical change in an analysis sample due to the action of the enzyme, and converts it into an electrical signal. In the biosensor, L-glutamate oxidase (hereinafter referred to as the "enzyme of the present invention") has extremely high substrate specificity for L-glutamic acid as an enzyme, does not substantially act on amino acids other than L-glutamic acid, and has high stability. The present invention provides a biosensor characterized in that it uses a biosensor.

Effects Unlike known biosensors for L-glutamic acid analysis, the biosensor of the present invention is easy to manufacture because it uses oxidase, which is the most commonly used receptor part of biosensors. Furthermore, the means for detecting the enzymatic reaction is selected depending on the purpose from among known means, such as means for detecting a decrease in oxygen, which is a substrate, or means for detecting an increase in hydrogen peroxide or ammonia, which are reaction products. It can be done and has variety. Furthermore, compared to known biosensors, the biosensor of the present invention has a lower L
- Specificity for glutamic acid is extremely high, and even in samples containing many types of amino acids, only L-glutamic acid can be selectively analyzed without being interfered with by other amino acids.

By using the biosensor of the present invention as described above, it is not only possible to selectively analyze L-glutamic acid in analytical samples that contain L-glutamic acid as an original component, such as foods and fermentation liquids, but also to analyze L-glutamic acid selectively. - Participates in enzyme activity or reaction in reaction systems involving glutamic acid, such as glutaminase, glutamate racemase, glutamate-oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT), etc. Other substances can also be analyzed.

[■] Specific Description of the Invention 1) Biosensor In the present invention, the term "biosensor" refers to an enzyme that serves as a receptor for identifying a substance to be detected and detects chemical or physical changes in an analysis sample due to the action of the enzyme. It is composed of a transducer part, which is a signal conversion part that converts it into an electrical signal, and measures the electrical signal as a change in current or a change in potential, and analyzes the amount or presence of the substance to be detected in the analysis sample. means a device for (
[Region of Chemistry, Special Issue No. 134, Biomaterial Science Volume 1” pp. 69-79. April 1982, 20I
(See Nankodo Publishing and Kagaku Kogyo, June 1982 issue, pp. 491-496).

As mentioned above, a biosensor that uses an enzyme as a receptor is also called an "enzyme sensor," and an enzyme sensor that uses an electrochemical device as a transducer is also called an "enzyme electrode.""EnzymeElectrode", edited by Shu Suzuki.

pp, 65-106. November 1, 1981, ■Published by Kodansha and "Chemistry Reihei" Volume 36, No. 5.

pp. 343-849 (1982)). Also,
Enzyme sensors include an electrode-attached enzyme electrode in which a receptor part and a transducer part are in close contact with each other or in close proximity, and a reactor-type enzyme sensor in which a receptor part and a transducer part are separated.

The biosensor according to the present invention constitutes a novel biosensor by using a specific L-glutamate oxidase, which is a new enzyme, as the receptor part in the above-mentioned known biosensor, and achieves a remarkable effect compared to the conventional technology. It was realized by Therefore, in the present invention, other than the use of the enzyme of the present invention, known techniques can be employed, and furthermore, techniques to be developed in the future can also be employed as long as they meet the purpose of the present invention.

2) Enzyme of the present invention The enzyme of the present invention may be a highly stable L-glutamate oxidase that has extremely high substrate specificity for L-glutamic acid and substantially acts on amino acids other than L-glutamic acid. It does not matter its origin or origin, and its preparation method does not matter.

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 below are as follows.

(1) Action When the enzyme of the present invention uses L-glutamic acid as a substrate, 1 m
It requires 1 mol of oxygen and 1 mol of water and produces 1 mol of α-ketoglutaric acid, 1 mol of ammonia and 1 mol of hydrogen peroxide.

(2) Substrate specificity Table 1 shows the results of allowing purified preparations of the enzyme of the present invention to act on various amino acids. The concentration of each substrate was 10 mM, and the reactions were performed at pH 7.4 (0.1 M potassium phosphate buffer) and pH 6.0 (0.1 M 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.

As described above, 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 (0.6%) on L-aspartic acid in
), but has virtually no activity against other L-amino acids including L-glutamic acid and L-histidine, and D-glutamic acid, and at pH 6.0, L-
- Shows virtually no activity against aspartic acid.

In contrast to the enzyme of the present invention, the known enzyme is L-
The enzyme shows no activity (less than 0.1%) against aspartic acid, but 8.4% activity against L-glutamine and 68% activity against L-histidine. Different.

The km value of the enzyme of the present invention for L-glutamic acid is 2.1 x 10-4 M at pH 7.4, and the km value for L-aspartic acid is 2.9 x at pH 7.4.
It is 10-2M.

(8) Measurement of titer The titer of the enzyme of the present invention was measured by an oxygen electrode method. That is, 0 containing 10 mM sodium L-glutamate.
．． 1 ml of 1M potassium phosphate buffer (pH 7.4) was placed in an oxygen electrode cell, 10 μl of enzyme solution was added, and the oxygen consumption rate was measured. The amount of enzyme that consumes 1 μmol of oxygen for 1 minute at 30°C is defined as 1 unit (hereinafter referred to as “Unit”).
It is 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
), 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 (pH 5.0) is added to the reaction stop solution.
) and 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) at 50°C.
After incubating for 0 minutes, cool to room temperature and
The absorbance at nm is measured, and the amount of α-ketoglutaric acid produced is determined from the calibration curve.

(4) Optimal pH As shown in FIG. 1, the optimal pH is around pH 7 to 8.5. Measurement of enzyme activity at each pH was performed using L as a substrate.
- using sodium glutamate and 0.0% as buffer;
2M acetate buffer (pH 8.5-6.0), 0.2M potassium phosphate buffer (pH 6.0-85) and 0.2M
Glycine-sodium chloride-sodium hydroxide buffer (
pH 8.5 to 12.0) was used, and the test was carried out at 30°C.

In addition, in FIG. 1, the enzyme of the present invention (solid line) and the known enzyme (dotted line) are quoted from FIG. 1 of JP-A-57-48685 for comparison of the optimum pH of the enzyme of the present invention and the known enzyme. ) is also shown.

As is clear from FIG. 1, the enzyme of the present invention is different from known enzymes also in its optimum pH.

In addition, when aspartic acid is used as a substrate, the effective pH range is narrow, and the optimum pH is 7 to 8, but it has almost no effect on L-aspartic acid at pH 6.0 or below and pH 10.0 or above (pH 6. .01% or less of the activity towards glutamic acid).

(5) pH stability After holding at each pH of 3.5 to 11.5 at 37°C for 60 minutes, 45°C for 15 minutes, and 60°C for 15 minutes, enzyme activity against glutamic acid at pH 7.4 was measured.

As a result, under the conditions of holding at 37℃ for 60 minutes, the pH was 55~
It is stable in the range of 10.5 (Fig. 2).

solid line), pH 5.5 to 45°C and held for 15 minutes
It is stable in the range of 9.5 (Figure 3), 60℃,
Under the condition of holding for 15 minutes, it was stable in the pH range of 5.5 to 75 (Figure 4).

In addition, in order to compare the pH stability of the enzyme of the present invention and the known enzyme, FIG.
Quoted from Figure 2 of Publication No. 8685 and indicated by dotted lines. )
are shown together with those of the enzyme of the present invention.

As is clear from Figures 2 to 4 above, when comparing the stable pH ranges of the enzyme of the present invention and the known enzyme, the two are clearly different, with the former being more stable in a wider pH range than the latter. .

(6) Suitable temperature range for action A reaction was carried out for 20 minutes using sodium L-glutamate as a substrate at each temperature of 30° C. to 80° C., and the enzyme activity was measured using the MBTH method described above.

As a result, the optimal temperature range for the action of the enzyme of the present invention is 30 to 60°C.
The optimum temperature for action was around 50°C (Figure 5).
.

(7) Thermal stability: pH 5.5, pH 7.
After holding for 15 minutes under each condition of pH 5 and pH 9.5, p
Enzyme activity toward glutamic acid was measured in H7.4.

As a result, it is stable up to 65°C at pH 5.5, and shows approximately 50% residual activity at 85°C (Figure 6, ▲-▲
). At pH 7.5, it is stable up to 50°C, and 75
It shows a residual activity of about 60% at ℃ (Fig. 6, ●-●). p
Even in H9.5, it is stable up to 45°C and shows about 50% residual activity at 70°C (Fig. 6, ■-■).

In addition, in order to compare the stability of the enzyme of the present invention and the known enzyme,
The temperature stability curve of the known enzyme (quoted from FIG. 3 of Japanese Unexamined Patent Publication No. 57-43685 and indicated by a dotted line) is shown together with that of the enzyme of the present invention.

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

(8) Inhibition, Activation, and Stabilization In order to examine the effects of various additive substances on the enzyme of the present invention, a reaction solution containing 1 mM of each substance shown in Table 2 (pH 7.
4) The enzymatic reaction was carried out inside.

The results are shown in Table 2.

1) EDTA: ethylenediaminetetraacetic acid 2) NEM: N
-Ethylmaleimide 3) PCMB: parachloromercurybenzoate 4)
DDTC; diethyldithiocarbamate 5) Tyrone: 4
．． Disodium 5-dehydroxy-m-benzenedisulfonate As is clear from Table 2, the enzyme of the present invention is inhibited by about 45% by parachloromercury benzoate;
It is not inhibited at all by cupric chloride and diethyldithiocarbamate. On the other hand, known enzymes are completely inhibited by cupric chloride and diethyldithiocarbamate. However, the two enzymes also differ in how they are affected by inhibitors.

Note that an activator and stabilizer for the enzyme of the present invention have not yet been found.

(9) MaX with ultraviolet absorption spectrum (see Figure 7):
273 nm, 385 nm, 465 nm shoulder: around 290 nm, around 490 nm (10) Coenzyme The enzyme of the present invention is heat-treated or treated with trichloroacetic acid (TCA), and the supernatant obtained by centrifugation shows that its absorption is 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.

Also, from the Rf value in thin layer chromatography, F
It was identified as AD.

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

(11) The enzyme of the present invention purified by polyacrylamide gel electrophoresis showed a single band.

(12) Molecular Weight The molecular weight of the enzyme of the present invention was determined to be 135.000±10.000 by gel filtration using Sephadex G-200 (manufactured by Pharmacia Fine Chemicals).

(13) The pl was 6.2 when measured by isoelectric focusing using isofocusing ampholine (manufactured by LKB).

(14) Crystal structure and elemental analysis Since the enzyme of the present invention has not been crystallized, it has not been measured.

(15) Purification method Purification of the enzyme of the present invention can be carried out by appropriately combining salting-out method, isoelectric precipitation method, precipitation method with organic solvent, adsorption method with diatomaceous earth, activated carbon, etc., various chromatography methods, etc. can. 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 for producing the enzyme of the present invention belong to the genus Streptomyces and have the ability to produce the enzyme of the present invention.

A specific example of such a microorganism is X.
-119-6 strain can be mentioned. The mycological properties of this strain are described below.

A. Microscopic observation: Aerial hyphae are straight, with a width of 09 to 1.0 microns, and show simple branching. The sporophyte consists of a chain of many spores, forming a spiral with 2 to 5 turns.

The spores are slightly circular in shape, and the size is 09 to 1.0 x 1.
1×l, 2μ, and the 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 (cultured 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 slightly bulge as they penetrate into 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 Its growth is good. The basal hyphae are white-yellow.

Aerial mycelia are white, abundant, and carry gray spores.

No pigment formation was observed in the medium.

(6) Nutrient agar medium The growth is extremely good. The basal hyphae are whitish-yellow and swell as they invade the agar. 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 mycelia are white, abundant, and carry gray spores.

No pigment formation was observed in the medium.

C. The growth temperature range for physiological properties is 8 to 40°C, with the optimal temperature being 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 the Pridham-Godelive agar medium is shown in Table 3.

The above properties are summarized as follows. That is,
The aerial hyphae are spiral-shaped, and the surface of the spores is spiny. 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, Bergey's Manual of Determinative Bacteriology was used.
of Determinative Bacteriol
When this strain was identified according to the classification system in the 8th edition (1974) of Streptomyces, this strain belonged to the genus Streptomyces, but no known species that fully matched the characteristics of this strain was found. identified this strain as a new strain, and identified it as Streptomyces sp.
plomycesssp. 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 the deposit was made on June 5, 1980, and the deposit number was Research report No. 6560 (FERMP-65
60) 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 methods, such as physical treatments such as ultraviolet rays, X-rays, and γ-ray irradiation, and chemical treatments using drugs such as nitronoguanidine. Any highly productive mutant strain of the enzyme of the present invention obtained by mutating the enzyme can be suitably used. Furthermore, the purpose of the method for producing the enzyme of the present invention is basically to utilize the ability of the microorganism to synthesize an enzyme protein using the gene deoxyribonucleic acid (DNA), which carries genetic information related to the production of the enzyme of the present invention. Therefore, such genetic DNA can be inserted into an appropriate vector and introduced into microorganisms of other genera by transformation, or the genetic DNA can be introduced into microorganisms of other genera by cell fusion using the protoplast method. A method for producing the enzyme of the present invention using microorganisms obtained by manipulative techniques is also applicable.

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 wheat bran, defatted soybeans, rice bran, corn, rapeseed meal, wheat, rice, rice husks, etc. alone or in combination of two or more, and if necessary, the present invention. Nutrient sources that can be assimilated by the microorganisms used, such as glucose, sucrose, arabinose, fructose, mannitol, inositol, and 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 , salts such as calcium salts, manganese salts, magnesium salts, zinc salts, iron salts, phosphates, sulfates, etc., growth factors such as thiamine, riboflavin, nicotinic acid, pantothenic acid, biotin, p-aminobenzoic acid, and vitamin B12. These include a culture medium to which these substances have been appropriately added, or a culture medium in which these substances have been granulated in an appropriate formulation, 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, culture methods other than those described above, such as absorbing or coating a liquid medium on a suitable carrier such as a sponge, may be used.
679), a method of inoculating and culturing seed bacteria can also be employed as long as the microorganisms used can propagate and produce the enzyme of the present invention well.

The culture conditions are not particularly limited, as long as the conditions optimal for enzyme production are selected depending on the type of microorganism used. Usually, for example, 20-30°C. What is necessary is just to culture|cultivate for 5 to 15 days at pH5-7.

Collection of Enzyme of the Invention Use The enzyme of the invention produced by culturing microorganisms can be extracted and separated from the culture, that is, the culture medium and/or the cultured bacterial 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.

3) Receptor part In order to use the above-mentioned enzyme of the present invention as a receptor part of a biosensor, the enzyme of the present invention and the substance to be detected, L.
- The receptor portion must be constructed in such a way that contact with glutamic acid can be easily carried out, the enzyme reaction can proceed smoothly, and the enzyme of the present invention is not substantially eluted into the analysis sample solution. Therefore, the enzyme of the present invention is usually immobilized,
or semipermeable membranes (e.g., dialysis membranes, ultrafiltration membranes, etc.)
It is used coated with.

Immobilization is not particularly limited, as long as it is done in a manner that is compatible with the purpose of use of the receptor in a biosensor, and in such a manner that the enzyme of the present invention is not substantially deactivated and the enzymatic reaction is not inhibited. Converting enzymes”. Partly edited by Chibata,
Published by Gi Kodansha on March 20, 1975, and see "Ion Electrodes and Enzyme Electrodes" (cited above, 65-77).

In other words, the immobilization method used for the receptor moiety is the inclusion method (lattice type, mylocapsule type, etc.), carrier bonding method (covalent bonding method, ionic bonding method, physical adsorption method, etc.), crosslinking method, etc. A suitable method for preparing the immobilized enzyme may be selected. Specifically, carrier or support materials include cellulose (cellophane, filter paper, etc.), acetylcellulose derivatives, nitrocellulose (
collodion, etc.), various ion-exchanged cellulose derivatives (
DEAE-cellulose, TEAE-cellulose, ECT
Cellulose derivatives such as EOLA-cellulose, CM-cellulose, P-cellulose, etc.), starch, dextran derivatives (Sephadex (trade name), etc.), agarose, mannan (konjac mannan, etc.), chitosan, carrageenan, alginic acid, xanthan gum, Polysaccharides such as agar, collagen, gelatin, fibroin,
Proteins such as keratin, albumin, and gluten, polymeric gels such as polyacrylamide, polyvinylpyrrolidone, and polyvinyl alcohol, polyvinyl chloride, polymethyl methacrylate, polyacrylonitrile, polystyrene, polyaminostyrene, polyethylene, polypropylene, polyurethane, polycarbonate, and polyamide ( nylon, polyamino acids, etc.), fluoroplastics (Teflon (
(product name), silicone resin, adsorption resin, ion exchange resin, and other synthetic organic polymer materials, glass,
Inorganic substances such as silica, ceramic, alumina, kaolinite, bentonite, calcium phosphate, hydroxyapatite, and activated carbon are used as they are, or by introducing functional groups reactive with enzyme proteins into them, or by activating the functional groups. Ru. When immobilizing by the entrapment method, enzymes are incorporated into the lattice of gels such as polymer gels, cellulose derivatives, polysaccharides, and proteins, or they are immobilized using organic polymer substances, cellulose derivatives, etc. Enzymes can be coated with a semipermeable membrane to prepare microcapsules. In the case of a carrier binding method, a method of covalently bonding to an appropriate carrier by a diazo method, a pebuzide method, an alkylation method, or a crosslinking reagent (glutaraldehyde, hexamethylene diisocyanate, etc.), a carrier having an ion exchange group It can be immobilized by a method of ionic bonding or a method of physical adsorption.

In addition, when using a crosslinking method, a reagent having two or more functional groups, such as glutaraldehyde, isocyanate derivatives (hexamethylene diisocyanate, toluene diisocyanate, etc.), isothiocyanate derivatives (hexamethylene diisocyanate, etc.) Oceanato, etc.), N
, N'-ethylene bismaleimide, N, N'-(1
．． 2-phenylene) bismaleimide, N, N'-(
Immobilization can be achieved by crosslinking between enzymes using 1,4-phenylene) bismaleimide, N,N-polymethylene biiodoacetamide, or the like. An immobilized enzyme can also be prepared by combining two or more of these methods. By the above method, the enzyme of the present invention can be immobilized and prepared into an appropriate form such as film, gel, granule, chip, powder, microcapsule, tube, fiber, or container. The manner in which the obtained immobilized enzyme is used as a receptor part of a biosensor is as follows. In other words, in the case of an electrode-mounted enzyme electrode, an immobilized enzyme in the form of a membrane, gel, powder, or microcapsule is usually mounted in close contact with or in close proximity to the working surface of the measurement electrode, and more preferably a porous membrane permeable to the substrate. membrane (for example,
dialysis membrane, ultrafiltration membrane, laminated membrane, heterogeneous membrane, asymmetric membrane, etc.). The electrode membrane, the immobilized enzyme layer, and the coating membrane in contact with the sample solution may be a laminated membrane or a heterogeneous membrane in which all of them are integrated, or a laminated membrane or a heterogeneous membrane in which the first two or the latter two are integrated. It may also be configured as a heterogeneous membrane. In addition, in the case of a reactor-type enzyme sensor, a column is usually filled with a granular or powdered immobilized enzyme, or a reactor is configured with a tube-shaped or fibrous (holofiber-shaped) immobilized enzyme.
Can be used as a receptor part.

4) Transducer part The transducer part is the part that oxidizes the substrate in the analysis sample by the enzyme of the present invention in the receptor part, detects the chemical or physical change caused by this reaction, and converts it into an electrical signal. It is. In the present invention, a case where a chemical change is detected is exemplified, or a physical change, such as a heat balance, may be detected using a thermistor or the like.

The chemical changes accompanying the reaction by the enzyme of the present invention are a decrease in oxygen and L-glutamic acid and an increase in hydrogen peroxide, ammonia and α-ketoglutarate in the analysis sample, which are detected by the transducer section. This is usually an increase in oxygen absorption, an increase in hydrogen peroxide, or an increase in ammonia.

An oxygen electrode is used as a transducer that detects absorption of oxygen and converts it into an electrical signal.

Commonly used oxygen electrodes use platinum, gold, or
It has a basic structure in which a noble metal electrode such as iridium is used, an electrode made of silver, silver/silver chloride thread, saturated calomel, lead, zinc, aluminum, etc. is used as a reference electrode, and an electrolyte is interposed between these two electrodes. Specifically, the above-mentioned two electrodes and an electrolyte (an alkaline solution such as potassium hydroxide or sodium hydroxide) are integrated, and the working electrode is made of an oxygen-permeable electrode film (fused film; Teflon film, polypropylene film, polyethylene film, etc.). A composite electrode (diaphragm oxygen electrode such as a Clark electrode) that is configured to come in contact with the sample solution through a diaphragm or a separate electrode that has a working electrode and a reference electrode connected by a salt bridge is used. . The method may be a polarographic method or a galvanic cell method.

A hydrogen peroxide sensing electrode is used as a transducer that detects hydrogen peroxide and converts it into an electrical signal. Specifically, an electrode having a structure similar to that of the oxygen electrode described above may be used. For example, a Clark-type hydrogen peroxide electrode comprising a working electrode made of a noble metal electrode such as platinum, a reference electrode made of a silver electrode, an electrolyte and a hydrogen peroxide permeable diaphragm is suitable. In addition, by using a hydrogen peroxide decomposition catalyst such as peroxidase or molybdate together with the enzyme of the present invention as a receptor part, hydrogen peroxide and iodine ions are reacted, and a decrease in the activity of iodine ions is detected. It can also detect hydrogen peroxide,
In this case, an iodine ion electrode can be used.

As a transducer that detects ammonia and converts it into an electrical signal, a cation-selective electrode can be used when analyzing ammonia as ammonium ions, and a hydrophobic gas-permeable electrode can be used when analyzing ammonia as ammonia gas. An ammonia gas electrode consisting of a glass electrode fitted with a membrane can also be used.

The electrode-mounted enzyme electrode according to the present invention is constructed by mounting the enzyme of the present invention as the receptor portion on the electrode sensitive material permeable membrane of the various electrodes exemplified above or integrally with such a membrane.

Furthermore, the reactor-type enzyme electrode according to the present invention is composed of the above-mentioned various electrodes which are installed separately from the enzyme reaction part and the receptor part on which the enzyme of the present invention is immobilized or the actor.

Note that the present invention is not limited to the transducer sensing substance and transducer method.

5) Specific Examples Below, as an example of the biosensor according to the present invention, an example of manufacturing an electrode-mounted enzyme electrode will be shown as an example, and an example of the use of this electrode will be shown. 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.

Example 1 0.1 ml of L-glutami/acid oxidase (448 U/ml) solution was placed on a 30 mm x 30 mm square cellophane membrane (
0.1 ml of 2.5% glutaraldehyde solution was infiltrated into this, dried overnight at 4°C, and washed with water until no absorption substance at 280 nm was eluted. , L-glutamic acid oxidase-immobilized cellophane membrane was obtained.

The above immobilized enzyme membrane was cut into small circular pieces, and an oxygen permeable membrane (Teflon membrane,
A cellulose dialysis membrane (membrane thickness 50 um, manufactured by Wiesking) was further attached to the immobilized enzyme membrane to produce an L-glutamic acid sensor (Figure 8).

In Figure 8, the diaphragm oxygen electrode has a platinum electrode (1) as the cathode, a lead electrode (2) as the anode, potassium hydroxide solution as the internal solution (electrolyte (3)), and oxygen on the surface of the platinum cathode. A permeable Teflon membrane (4) is attached.The above-mentioned immobilized enzyme membrane (5) is attached on this Teflon membrane,
Furthermore, it is covered with a cellulose dialysis membrane (6) and fixed with an O-ring (7).

Using the above L-glutamic acid sensor, an L-glutamic acid analyzer (not shown in FIG. 9) was manufactured.

In FIG. 9, the sample solution (2) injected from the sample solution inlet (1) is transferred to the flow cell (5) together with the dissolved oxygen saturated buffer (3) by the peristaltic pump (4).
). At this time, the L-glutamate sensor (
The current reduction value measured by 6) is recorded on the recorder (7).

A sodium L-glutamate standard solution was continuously injected into the above device, the current reduction value was measured, and the correlation with the sodium L-glutamate concentration was determined. The results are shown in FIG.

Using the same device, the response at each temperature was measured, and the results are shown in FIG. However, the measurement was 0.8mML-
Using sodium glutamate solution, 20℃, 30℃
and 40°C.

Similarly, sodium L-glutamate solutions at each pH (0
．． 8mM) was measured (at 30°C), and the results are shown in Figure 12.

In addition, the responsiveness to various amino acids was similarly measured, and the results are shown in Table 4. Note that the concentration of each amino acid solution is 1
．． 0mM, and the reaction was carried out at pH 7.0 (0.1M potassium phosphate buffer).

Example 2 0.5 ml of L-glutamate oxidase (12.5 U/ml) solution was dropped onto a porous nitrocellulose membrane (manufactured by Toyo Kagaku Sangyo ■, TM-5; pore size 0.1 um, diameter 25 mm, film thickness 140 μm). - Adsorption and immobilization was performed by suction to obtain an L-glutamate oxidase-immobilized membrane.

The immobilized enzyme membrane was cut into a circular shape (5.0 mm in diameter), and then attached to a diaphragm oxygen electrode in the same manner as in Example 1 to produce an L-glutamic acid sensor.

Usage example Commercially available soy sauce is diluted with 0.1M phosphate buffer (pH 7.0) for 10 minutes.
A sample solution was prepared by diluting the solution twice and bubbling air into it to saturate it with dissolved oxygen. 1 ml of the above sample solution was injected into 49 ml of 0.1M phosphate buffer (pH 7.0), and the measurement was performed using the apparatus manufactured in Example 1. The concentration of L-glutamic acid in the soy sauce was calculated from the obtained current reduction value using a calibration curve. Glutamate autoanalyzer (Technicon)
The results were in good agreement with the quantitative values determined by Innostoulments Co., Ltd.).

Reference example 20g of wheat bran and 16g of water in a 500ml Erlenmeyer flask
Streptomyces sp.
-6 (Feikoken Bacteria No. 6560) was inoculated, and the temperature was 28°C. A seed culture was prepared by culturing for 7 days.

Put 200 g of wheat bran and 160 ml of water in each of 25 5-liter Erlenmeyer flasks, autoclave at 120°C for 30 minutes, inoculate the above seed culture aseptically, and culture at 28°C for 2 days, then raise the temperature to 20°C. The cells were lowered and cultured for an additional two weeks.

After soaking the obtained culture in 37.5 liters of water for 1 hour,
Filter and further pass through diatomaceous earth to obtain crude enzyme liquid 34
I got l. Ammonium sulfate was added to this crude enzyme solution to saturation of 50%, and the resulting precipitate was collected by centrifugation, dissolved in 3.9 liters of 0.02M acetate buffer (pH 5.5), and incubated at 57°C for 3.9 liters.
Heated for 0 minutes. After cooling this heat-treated enzyme solution to 5°C or below, double the volume of pre-chilled ethanol was added, and the precipitate formed was collected by centrifugation and added to 0.02M phosphate buffer (pH 7).
．． 4) Dissolved in 2 liters and dialyzed against the same buffer overnight. The precipitate generated during dialysis was removed by centrifugation, and the supernatant was passed through a DEAE (diethylamine ethyl)-cellulose column (3.5 x 50α) equilibrated with the same buffer to remove the adsorbed enzyme. Elution was performed using the same buffer. The eluted active fraction was collected and 0.05M containing 0.05M NaCl
Dialysis was performed overnight against M acetate buffer (pH 55). This dialysate solution was equilibrated with the same buffer solution to DEAE-Sepharose C.
The mixture was passed through a L-6-13 (Falman Fine Chemicals) column (2 x 10 cm), and the adsorbed enzyme was eluted using a linear gradient method using 0.05 to 075 M sodium chloride. The eluted active fraction was collected, dialysis concentrated, gel filtration was performed using a Cephadex G-200 (manufactured by Pharmacia Fine Chemicals Co., Ltd.) column (2.5 x 120 cm), the active fraction was collected, and after concentration, 0 Dialysis was performed against .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 protein.

A yield of 184%) was obtained.

[Brief explanation of drawings]

FIG. 1 shows the action pH ranges of the enzyme of the present invention (solid line) and the known enzyme (dotted line). FIG. 2 shows the stable pH range (held at 37° C. for 60 minutes) of the enzyme of the present invention (solid line) and the known enzyme (dotted line). 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 of the enzyme of the present invention (60°C for 15 minutes)
shows. 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, ○−○
) indicates the stable temperature range. In Figure 6, ▲-▲ is the pH
5.5, ●-● indicates the temperature stability curve under each condition of pH 7.5, and ■-■ indicates pH 9.5. FIG. 7 shows the ultraviolet absorption spectrum of the enzyme of the present invention. FIG. 8 schematically shows the L-glutamate sensor according to the present invention. 1...Platinum electrode, 2...Lead electrode, 4...Teflon membrane 5...Immobilized enzyme, 6...Cellulose dialysis membrane Figure 9 is L- in Figure 8 This figure schematically shows an L-glutamic acid analyzer using a glutamic acid sensor. 2...Sample solution, 3...Dissolved oxygen saturated buffer 5
...Flow cell, 6. L-glutamate sensor, 7. Recorder. Figure 10 shows the correlation between sodium L-glutamate concentration and current reduction value when using the device shown in Figure 9. It shows. FIG. 11 shows the response at each temperature when the device shown in FIG. 9 is used. FIG. 12 shows the responsiveness at each pH when the apparatus shown in FIG. 9 is used. Patent applicant (6.77) Yamasa Shionura Co., Ltd. Figure 1 Figure 2 Figure 5 Figure 6 Figure 6 Temperature Figure 7 No δJ b 9tB Chi-LFL (1 house 1) Sillua (phantom 4, t471! De lθ〃 P〃 Procedural amendment (voluntary) March 1980 Noah El 1, Indication of fact II 1988 Patent Application No. 146707 2, Name of the invention Biosensor 3, Relationship with the person making the amendment case Patent applicant address (zip code 288) 2-10 Shinseicho, Choshi City, Chiba Prefecture 1 Telephone 0479 (
22) 0095 4. Column for detailed description of the invention in the specification to be amended, column for simple explanation of the drawings, and drawing 5. Contents of the amendment. The one that says free [■I7-glutamic acid decarboquinola-
Enoelina coli 1 and dl'1 with seven activities. 2) Specification P) In the 3rd line of page 3, it says 1, which is 1 potassium ion, which exists in Daikichi, but it is corrected to 11, such as 1-potassium ion, exists in the measurement system.'' 3) In the 8th line of page 10 of Specification 111, the word "Practice" is corrected to; Completion 1. 4) In Specification 7J1, page 11, line 1, ``enzyme'' and 1- are listed, and 1- is between 1 and 1 (hereinafter referred to as 1 enzyme).''
join. 5) In the following sections (1) to (8B) of the specification, all references to ``Enzyme J of the present invention are corrected to 1 enzyme 1.'' (1) Page 12, line 6, (2) End of page 12 line, (3) 141'↓9th line from the bottom, (4) last line of page 14, (5)
Page 15, line 6, (6) Page 15, line 11, (7) 1st
Page 6, line 5, (8) Page 17, line 3, (9) Page 17, line 4, +101 171'↓ Line 7, +113 Page 18, line 6, (1'2 Page 18 Line 9, (13) page 18, line 1I, +14) page 18, line 19ti, 05) page 19, line 14, +161 page 19, line 21, (19) line 21
page 1st line, (20) page 21, line 8, 31) page 21, line 14, (4 page 21, line 18, (a)) page 22, line 2
line, (24) page 22, line 6, (branch)) page 22, line 9, (branch)) page 22, line 18, (Jun page 23, line 1, ρ
88 No. 24, line 5, C1, page 24, lines 6-7, Suzu 0), page 29, line 6, Town), page 29, line 10, and (82, page 29, line 13. 6) Details Delete -1 of 1 of the present invention on page 29, line 5 of the book. 7) Page 29, line 12 of the specification] The enzyme of the present invention is basically replaced with ``Furthermore, the production of the enzyme by the microorganism is basically,''g]', ilF, do. 8) Specification F "Page 29, line 15, there is something Iζ to do. The word 1 is to be 1. Correct RJ to -1. 9) Specification, page 30, lines 1-2. 10) Specification U (Delete -1 for 1 of the present invention on page 30, line 14. +1) 1 unit on page 37, lines 12 and 13 of specification H11.
1 (membrane-like, membranous, fibrous, or porous fibrous)
and iJ pressure. 12) Specification, 11, page 43, lines 7 to 9.1 Note that each amino acid was used. ''In addition, for amino acids other than L-glutamic acid, use a mil'i of 2.01 nM61, and for L-glutamic acid, use a 0.4 mM solution.
Ogawa was measured and the measured values were converted to calculate the relative response value. The reaction is I) H5.5(0.], ]lvl acetic acid-10'
1 acid-j-1 lium buffer). 13) Details; 1: Revised Table 4 on page 43 as follows 1'
do. X'- \, 1 ------- Table 4 14) Between line 10 and line 1I on page 44 of the specification, written down as Example 3 after the description of Example 2. Add the text. 1 Example 3 The L-lyrutamic acid oxidase-immobilized cellophane membrane obtained by the same operation as in Example 1 was cut into small circular pieces, and placed on a hydrogen peroxide electrode (newly manufactured by Ishikawa Seisakusho). The immobilized enzyme membrane was then covered with a cellulose dialysis membrane to produce an L-glutamate sensor having the same structure as in Example 1. Using this sensor, L
- Manufactured a glutamic acid analyzer. Four consecutive standard solutions of sodium L-glutamate were introduced into the above apparatus, the current value was measured, and the correlation with the concentration of sodium L-glutamate was determined. The results are shown in FIG. 15) Details: 1: Add the following sentence after the description of the usage example up to the third line of page 45. 1-Also, the known ITI of sodium L-glutamate: (5-]001tmoIe/Hl) was added to commercially available sauce n11, and the measurement was carried out using the apparatus manufactured in Example 1 and Example 3. When the concentration of 17-glutamic acid in soy sauce was calculated from the calibration curve, a measured value almost proportional to that for addition was obtained. The relationship between the measured value and the amount added is shown in Figure 14 (
(using the apparatus of Example 1) and FIG. 15 (using the apparatus of Example 3). 116) Specification page 50 No. 13
13th to 15th line after the brief description of the drawing up to the line
Add the following text to explain the figure. [Figure 13 shows the correlation between the current value measured using the L-glutamic acid analyzer described in Example 3 and the concentration of sodium L-glutamate. FIG. 14 shows the correlation between the measured values obtained using the apparatus shown in FIG. 9 and the nails of L-glutamate added to soy sauce. FIG. 15 shows the correlation between the measured values obtained using the L-glutamic acid analyzer described in Example 8 and the amount of L-glutamate added to the sample. ] 17) Attach a separate sheet of drawings (S13-15) as additional drawings to the application 11F; (i

Claims (1)

[Scope of Claims] 1) A biological system comprising an enzyme as a receptor for a substance to be detected and a transducer that detects a chemical or physical change in an analysis sample due to the action of the enzyme and converts it into an electrical signal. A biosensor characterized in that the sensor uses L-glutamate oxidase, which has extremely high substrate specificity for L-glutamic acid, does not substantially act on amino acids other than L-glutamic acid, and has high stability. 2) The biosensor according to claim 1, wherein the transducer section is an electrode that detects a chemical change in an analysis sample and converts it into an electrical signal. 3) The biosensor according to claim 1 or 2, wherein the receptor portion comprises an immobilized enzyme.