JPS6042910B2 - Quantification method of L-amino acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for quantifying L-amino acids using a microbial electrode. The treated ones were carbonic acid-sTEL, Sendou 1, LS
-Top 1■1'゛hj,,tsu! Concerning the quantitative method of amino acids.

As a method for quantifying L-amino acids, L-glutamic acid decarboxylase (L-glutamate decarboxylase) derived from pumpkin, Escherichia coli, or Clostridium welsh has traditionally been used.
matedecarboxylase) or L-lysine decarboxylase (L-lysine decarboxylase) from Escherichia coli or Bacterium cyadaveris.
box-ylase), the following reaction of formula (I):
L-amino L-7S'' Applicable amine + C02 (I
) The Warburg pressure method, which measures the amount of carbon dioxide gas generated by manometry, and the autoanalyzer method, which colorimetrically determines the amount of carbon dioxide gas generated, are known.

These are accurate and excellent methods, but because it is difficult to use enzymes continuously, expensive enzymes must be used anew each time a measurement is performed, making them uneconomical, and the autoanalyzer method In some cases, the equipment is complex and expensive, and enzyme adjustments are required.

Although it is possible to immobilize the enzyme and use it continuously, this method has not yet been put to practical use.
In view of the above-mentioned problems, the present inventors conducted research on a simple and accurate method for quantifying L-amino acids, and found that, for example, Escherichia spp., Citrobacter spp., Clostridium spp., Bacillus spp., Streptococcus spp., Pseudomonas spp., Bacillus spp. Active bacteria that belong to the genus Myxococcus, Lactobacillus, etc., ascomycetes such as Aspergillus, or yeasts such as Rhodotorula, and have a decarboxylase for the L-amino acid to be quantified. A microbial electrode, in which activated microbial cells whose bodies have been treated with an organic solvent and/or surfactant are attached to a dissolved carbon dioxide electrode, is immersed in a test solution containing the above-mentioned L-amino acids under anaerobic conditions, or We have discovered that by measuring the electromotive force of the microbial electrode generated by contact with the microorganism, it is possible to easily and accurately quantify the target L-amino acid whenever necessary, and have completed the present invention. It came to this.

The feature of the method of the present invention is that the reaction of formula (1) above is carried out using a specific enzyme system within the microorganism, and the CO2 produced is
In a method for quantifying gas with a carbon dioxide electrode, formula (1) is carried out under anaerobic conditions, and a microbial electrode is used in which active bacterial cells treated with an organic solvent and/or surfactant are attached to the carbon dioxide electrode. There is something to do.

The effect can be obtained by using untreated bacterial cells (Japanese Patent Application No. 53-3
5906 (Japanese Unexamined Patent Publication No. 54-128394).
1) Selectivity is improved because harmful enzymes other than the specific enzyme system are inactivated. (For example, when measuring L-glutamic acid using live cells of Escherichia coli, if L-glutamine coexists, glutaminase also coexists, so L-glutamic acid is produced, making it impossible to measure only L-glutamic acid. However, when acetone-treated cells are used, glutaminase is inactivated, so they no longer respond to L-glutamine.) In addition, (2) in the case of treated cells, the properties of the cell membrane are altered. (3) It is easier to produce than enzymes;
Important features include good stability of enzyme activity within the bacterial cells and improved storage stability. The microbial electrode using untreated bacterial cells disclosed in the prior invention relating to the method for quantifying L-glutamic acid and L-lysine has problems such as problems in selectivity, storage stability, and reproducibility depending on the type of bacterial cells. However, this problem was solved by the present invention.

The present invention will be explained below. The microorganism used in the present invention is, for example, used to quantify L-glutamic acid.
As a strain with strong Iase) activity, for example, Escherichia coli ATCC
873λ Citrobacter freundei (CitrOb
ATCClO787, Clostridium welsh (ClOStridil)
]Nlwelchii) ATCCl3l2tun Rhodotorula●glutinis (RhOdOtOrlJlaglutin
is) IFOO4L is used and L-lysine decarboxylase (L-lysine decarboxylase) is used to quantify L-lysine.
As a strain with strong rbOxylase activity, for example, Escherichia coli (Escherichia coli)
ATCC23226, Bacterium chyadabellis (B
acterium cadaveris) ATCC976
Hestreptococcus huaecalis (StreptO
cOccusfaecalls) ATCCl2984,
Pseudomonas ●Satsukarofia (PSeUdOmOr
klSsaccharOphia) ATCCl5946
, Bacillus subtilis
lls) ATCCl5O37, Myxococcus virescerls
) ATCC252O3, Clostridium welchii ATCC
l3l27k Lactobacillus casei (LactOba
cillus casei) ATCC7469, and Aspergillus niger (A) Pergllusnige.
r) ATCC6278, etc. are used, and L-phenylalanine decarboxylase (L-phenylalanine decarboxylase) is used to quantify L-phenylalanine.
Examples of strains with strong oxylase activity include Streptococcus faecalis (StrePtOCOCC).
LlSfaecalis) ATCC8O4 is used.

In addition, in order to quantify L-arginine, L-arginine decarboxylase (L-Ayginine decarbOx
As a strain with strong ylase activity, for example, Escherichia coli ATCC
IO787, etc. are used, and amino acids not listed here can also be quantified using microorganisms in which the corresponding decarboxylases exist.

The activated bacterial cells used in the present invention are prepared by culturing them in a normal nutrient medium suitable for each strain, washing them 2 to 3 times with pure water, and then using acetone, ethanol, methanol, isopropanol, n-propanol, etc. Organic solvents such as toluene and ether and/or cetyltrimethylammonium bromide (Cetyltrimethylammonium bromide)
mmOnillmbrOmide For example, “LissOl
amirleA” (ICI trademark)), cetylpyridinium bromide (CetylpyridiniLlmbr)
Omide), e.g. "FixanOlC" (ICI trademark), cetylpyridinium chloride (Cetylpyridinium chloride)
cationic surfactants such as (ridiniumchlOride), sodium-higher alcohols and sulfates,
For example, anionic surfactants such as "Silpit SP" (trademark of Ipposha Yushi Kogyo K.K.), polyoxyethylene alkyl ether (POlyOxyethylene
Alkyl●Ether) For example, Sanmorin January (Sanyo Yushi Kogyo K.K. trademark), polyoxyethylene●alkyl●phenol●ether (POlyOxyeUly
lene●Alkyl◆PherlOI●Ether)
For example, nonionic surfactants such as "Scoreol" (Kao Soap K.K. trademark) and highly permeable surfactants such as amphoteric surfactants such as alkyl betaine can be used to remove L-amino acids. After immersion treatment using a surfactant that does not inhibit carbonic enzyme activity,
Dry powder at low temperature.

If this powdered activated bacteria is stored in the freezer, its activity will be maintained for a long time (2 years or more).About 1 Cf/y of viable bacteria can be confirmed in these dry powders. . The microorganism electrode of the present invention is attached to the diaphragm of a conventional commercially available dissolved carbon dioxide electrode, as shown in Fig. 1, and is made by dissolving activated-treated bacterial cell powder of the above microorganism in water to form a paste. is coated on a carrier 3 such as a millibore filter paper piece or a nylon mesh, and covered with a membrane 4 having micro-pores that are impermeable to microorganisms, such as a cellophane membrane, and placed on the diaphragm 1 as shown in FIG. It can be easily created by attaching it.

Furthermore, it can be similarly prepared using immobilized microorganisms obtained by immobilizing microbial cells powdered as described above with collagen or acrylamide gel, for example. As the thin film 4 having micropores for covering the bacterial cell layer 2 of the microorganisms, any thin film 4 may be used as long as it does not pass through the bacterial cells of the microorganisms used in the present invention but allows carbon dioxide gas etc. to freely pass therethrough. For example, any membrane that satisfies the above conditions can be used, such as a porous membrane such as a millibore filter, a dialysis membrane such as cellophane, or an animal semipermeable membrane.

Note that when an immobilized microbial membrane is used, the above-mentioned thin film 4 is not necessary. In Figure 1, 1 is the diaphragm (silicone membrane) of the dissolved carbon dioxide electrode, 2 is the microorganism layer, 3 is the carrier, and 4 is the dialysis membrane (
cellophane membrane), 5 is the PH electrode, 6 is the internal liquid (NaHC
7 indicates the entire microbial electrode. When this microbial electrode is brought into contact with a test liquid such as a fermentation liquid in the presence of oxygen, the microorganisms near the diaphragm absorb nutrients (sugars, sugars, etc.) in the test liquid.
It is impossible to quantify amino acids such as L-glutamic acid and L-lysine because they assimilate and/or respire amino acids (amino acids) and release a large amount of carbon dioxide gas.

On the other hand, the present inventors have found that if the microbial electrode and the test solution are brought into contact with each other under anaerobic conditions, the production of carbon dioxide gas due to the life activity of the microorganisms mentioned above is completely suppressed, and therefore, the equation (1) is satisfied. As mentioned above, the present invention was completed based on the discovery that the reaction proceeds quantitatively. The set of quantitative system shown in FIG. 2 is one of the embodiments of the present invention. 7 in FIG. 2 is a microbial electrode;
9 is a flow cell, 8,8″ is a rubber backing, 10 is N
2 gas inlets, 11 a buffer solution inlet, 12 a sample inlet, 13 a PH meter or ion meter, and 14 a recorder.

The measuring method of the present invention will be explained below according to the system shown in FIG. First, a constant flow rate from the buffer inlet 11 is caused to flow into the flow cell 9 while blowing N2 gas through the inlet 10, and the electromotive force of the electrode is recorded on the recorder 14.

Inject samples from 12 to 1 to 30 with an injection time of 0.5 to 5 minutes.
Inject sequentially at minute intervals. This sample liquid is appropriately diluted with a buffer solution and reaches the flow cell 9. In the flow cell 9, the corresponding L-amino acid in the sample is decomposed by the enzyme of the microorganism by the reaction of formula (1), and CO2 gas is generated. This CO2 gas passes through the diaphragm 1 and changes the pH of the internal liquid. The PH change is measured by the PH meter or ion meter 13 via the PH electrode 5, and recorded on the recorder 14. = Ni = - Wealth ■ = Ni, and the Nernst equation (■) is established.

In the formula, EO is the asymmetric potential difference, R is the gas constant, T is the absolute temperature, and F is the Faraday constant. Using this formula (■), the substrate concentration of the test solution can be determined.

Regarding the conditions during measurement, the pH of the measurement was 3.5 to 6. \
The temperature is preferably in the range of 20 to 40°C, and the contact time between the sample and the electrode is 0.5 to 5 minutes, and usually reaches the saturation value in 3 minutes.

The measurement concentration range of the substrate is 10-1 to 10-4M, making it possible to measure a wide range, and the linearity of E(!1.10gC is very good. The buffer solution used is citric acid, Organic acid buffers such as fumaric acid and succinic acid, or pyridine-hydrochloric acid buffers are used.In particular, NaC
A pyridine-hydrochloric acid buffer containing KH2PO4 (0.5 y/dl each) and pyridoxal-5''-phosphate (0.1 V/1) is preferred.
In Figure 2, N2 gas is used to create an anaerobic condition, but it is not limited to N2 gas, and in short, it is fine as long as dissolved oxygen does not coexist, and it can be replaced with another inert gas. Also good,
Further, a carrier containing no dissolved oxygen may be used. When used under the above conditions, the activity is maintained for more than 3 weeks with continuous use. Since the method of the present invention uses actively treated microbial cells, it is more susceptible to impurities such as amino acids other than L-amino acids, organic acids, sugars, etc. that are to be quantified compared to prior art techniques that use pure enzymes. However, for example, when quantifying L-glutamic acid as an L-amino acid, P.
When measured using H4.4O at a temperature of 30 DEG C., as shown in Table 1 below, when L-glutamic acid is taken as 100%, no electromotive force was found that was 5% or more.

Other L-amino acids other than L-glutamic acid to be determined that are not shown in Table 1, namely L-methionine, L-histidine, L-lysine, L-proline, L-serine, L-isoleucine, L- -Phenylalanine, L-
Leucine, L-asparagine, L-valine, L-threonine, L-ornithine, L-citrulline and malic acid,
No effect was observed on pyruvic acid, glucose, urea, etc.

On the other hand, when active bacterial cells not treated with a solvent or a surfactant were used, the selectivity was 108% for L-glutamic acid compared to 100% for L-glutamic acid, which was a 27-fold increase in selectivity.

In addition, even if untreated bacterial cells exhibit no activity and cannot be measured, they can be activated and quantified by treatment with a solvent and/or surfactant, such as Citrobacter freundei (CitrObacter freundei).
urldii) In the case of ATCClO787, enzyme activity was hardly expressed for L-glutamic acid in toluene-untreated bacterial cells, making it impossible to measure L-glutamic acid, but it was possible to measure L-glutamic acid in toluene-treated bacterial cells. be.

In addition, in the Warburg manometry method using the same Escherichia coli, 45% L-tyrosine, 80% L-arginine, and 2 L-tryptophan were added to 100% L-glutamic acid.
5% and malic acid 40%.

In addition, when enzymes derived from pumpkin are used, the influence of urea is significant, making it impossible to measure L-glutamic acid fermentation and other L-amino acid fermentation processes that use urea as a nitrogen source.
On the other hand, it can be seen that the method of the present invention is extremely superior in terms of selectivity of the microbial electrode of the present invention. As mentioned above, the method of the present invention has (1) better selectivity and (2) better selectivity than conventional enzyme methods or methods using active bacterial cells.
This method is superior in terms of modification of the bacterial cell membrane, (3) preservation of the bacterial cell source, etc., and is easier and more accurate than conventional methods.
- Provides a method for quantifying amino acids.

In the examples, percentages indicating compositions indicate f/Dt% unless otherwise specified.

Example 1 Escherichia coli
) ATCC8739 was cultured in a shaking flask at 30°C using the medium shown in Table 2.

After culturing for 2 CH1, 50 ml of the culture solution was centrifuged to obtain wet bacterial cells.

Add this to 0.1M. ．． After washing twice with KC1 solution, it was suspended in 5 ml of water, 50 ml of acetone was added dropwise at room temperature, stirred for 5 to 10 minutes, and centrifuged under cooling. This was washed twice with acetone and once with ether, dried in a vacuum desiccator, and
．． 6y acetone/ether treated bacterial cells were obtained. 1 above
Dissolve ~2 m9 of acetone/ether-treated bacterial cells in a small amount of water to make a paste, spread it on a nylon mesh with a diameter of 107 wt, and use a cellophane membrane 4 to connect it to a dissolved carbon dioxide electrode (E5O3 diode) as shown in Figure 1. , Radiometer, Denmark).

Using this microbial electrode, a flow cell 9 (
A measurement system as shown in Fig. 2 was assembled by inserting it into a tube with a capacity of 0.5 mt through a rubber backing 8.8''.

As a buffer solution, 0.1M pyridine-hydrochloric acid buffer (0.5y/d1 NaCl and KH
2PO4) from 11 to 57rL1/Mi in Figure 2.
N2 gas is introduced at a flow rate of 10 to 0.21/M.
The electrode 7 was connected to a PH meter 13 and a recorder 14 to measure the electromotive force.

During the measurement, the temperature inside the flow cell 9 was maintained at 30°C. For the sample, a 4800 ppm L-glutamic acid aqueous solution and its diluted solution were sequentially injected from 12 at a rate of 1 Tnt/Min for an injection time of 3 minutes. As this sample was diluted with a buffer and flowed into the flow cell 9, the microbial electrode 7 began to give an indication, and after 3 minutes the indication reached a saturation level, and a peak as shown in FIG. 3 was recorded. Third
In the figure, the vertical axis shows the electromotive force MV of the electrode, and the horizontal axis shows time.
The relationship shown in FIG. 4 was observed between the height of each peak in FIG. 3 and the glutamic acid concentration of 10 gC in the flow cell, and its slope of 58rnV/PCcH almost matched the coefficient of the Nernst term in equation (2). (Theoretical value: 60.16n
1 V at 30°C) On the other hand, Frechbacterium lactofermentum ATCCl3869 was incubated at
Aerated agitation culture was performed at 0°C.

The obtained culture solution was diluted 2@ to prepare sample A, and a known amount of L-glutamic acid as a reagent was added to this to prepare sample B.
, C, and D were prepared.

The peak values of these samples were read according to the system shown in FIG. 2, and the concentration of L-glutamic acid was determined from a calibration line prepared using a standard concentration solution.

The results 1 were as shown in Table 4, and were in good agreement with the values measured by the conventional autoanalyzer method (using pumpkin enzyme) for each sample. Example 2 Clostridium Welsh (CIOstridium)
Mwelchll) ATCCl3l24 was cultured in a culture solution containing 3% trypsin-digested triglyceride, 2% glucose, and meat pieces at 30° C. for 2 cycles with shaking in a flask through hydrogen gas.

Using 50 ml of culture solution in the same manner as in Example 1, 0.3 f1
After obtaining acetone/ether-treated bacterial cells, a microbial electrode was constructed, and a sample of the L-glutamic acid fermentation liquid obtained in the same manner as in Example 1 was obtained over time.In the toluene-untreated bacterial cells, almost no enzyme activity was expressed. Therefore, it was not possible to measure L-glutamic acid. Example 4 Rhodotorula◆glutinis (RhOdOtOrula*A
, B, C, and D).

The results are shown in Table 5, and were in good agreement with the values measured by the conventional autoanalyzer method using pumpkin enzyme for each Sanra®. When microbial cells that had not been treated with acetone/ether were used, the values were considerably higher than those obtained by the conventional autoanalyzer method, and there was a problem in terms of selectivity for other L-amino acids. Example 3 Citrobacter Freundei
rfreurldll) ATCClO787 was cultured under the same conditions as in Example 1 using the medium composition shown in Table 2.

Centrifuging 50 ml of the culture solution to obtain wet bacterial cells, 0.1 M.
KC1 solution! After washing twice, the suspension was suspended in 5 ml of water, 5 ml of toluene was added dropwise at room temperature, stirred for 10 to 2 minutes, and centrifuged under cooling. This was dried in a vacuum desiccator to obtain 0.5 y of toluene-treated bacterial cells, which constituted a microbial electrode. Example 2
Regarding L-glutamic acid fermentation liquid of the same sample as L-
The glutamic acid concentration was determined and Table 6 was obtained. Culture solution 50ml
The wet bacterial cells obtained by centrifugation were diluted with 0.1 M. ．． After washing twice with KC1 solution, 20 m9 of cetyltrimethyl-ammonium bromide (Cetyltrimethyl-Ammonium bromide) was added.
The cells were suspended in 50 mt of an aqueous solution containing mOniumbrOmide), stirred for 5 to 10 minutes, and then treated with acetone and ether in the same manner as in Example 1 to obtain 0.6 q of bacterial cells.

A microbial electrode was constructed using this, and the glutamic acid concentration was determined for the L-glutamic acid fermentation liquid of the same sample as in Example 2, and it was found to be in good agreement with the value measured by the conventional autoanalyzer method using pumpkin enzyme. . In this case, as in Example 3, almost no enzyme activity was expressed in the bacterial cells that were not treated with the surfactant, making it impossible to quantify L-glutamic acid. Example 5 Lactobacillus casei
casei) ATCC7469<Test NO. l) with 10% skim milk powder, 0.5% L-lysine salt, and 1 % pyridoxal.
In a culture solution consisting of 00 μda/e, Streptococcus
Str'EptOcOccusfae
calls) ATCCl2984 (NO.2), Pseudomonas satsukalovia (PseudOmOnass
accharOphia) ATCCl5946 (NO
．． 3), Bacillus subtilis
btllls) ATCCl5O37 (NO.4), Myxococcus virescens (MyxOcOccusv
irescens) ATCC252O3 (NO.5) with 1% meat scratches, 1% polypeptone, NaClO. 5%,
L-lysine hydrochloride 0.5%, pyridoxal 100μf
In addition, Aspergillus niger (Aspergillus niger) ATCC627
8 (NO.6) with 2% glucose, KII2PO4O.
1% and koji juice supplemented with lime carbonate.After 2 hours, 50ml of each culture solution was centrifuged to obtain wet bacterial cells, each containing 0.1M, . After washing twice with KCI solution 5
The suspension was suspended in mt of water, and 5 ml of acetone was added dropwise at room temperature, followed by the same method as in Example 1 to obtain 0.5 to 0.6 y of acetone/ether treated microbial cells, respectively.

(This powder was cultured in a nutrient medium and viable bacteria of about 1 Cf/f was confirmed.) A microbial electrode was constructed using the method of Example 1 using the active bacteria treated with this solution, and the method of Example 2 was used. Regarding L-lysine fermentation liquids A, B, and C of similar samples, L-
When the lysine concentration was quantified, as shown in Table 8, it agreed well with the value obtained using the conventional acidic ninhydrin method. Example 6 StrePtOCOCCLlSfaecalls A
TCCl2984 was mixed with 3% trypsin-digested product of Tritin,
3 with a culture solution containing 1% glucose and 0.1% yeast autolysate.
rc Cultured for 115 hours and treated in the same manner as in Example 1 to obtain 0.5y of treated microbial cells, which were used to construct a microbial electrode. The following Table 9 was obtained by adding a certain amount of L-phenylalanine to samples A, B, and C, respectively, and comparing the measured values with the liquid chromatography method.

However, the buffer solution was the same as in Example 1, and the pH was 5.0.
The settings were used. The results showed good agreement between the two. Also, while the untreated bacteria remained active for only 1 month when stored in the refrigerator, the treated bacteria remained active for 6 months.
The activity was maintained. Example 7 Escherichia coli
) ATCClO787 was cultured in the same manner as in Example 6, and treated in the same manner as in Example 1 to obtain 0.3y of treated bacterial cells, which were used to construct a microbial electrode and L-glutamic acid as in Example 2. Different amounts of L-arginine were added to the fermentation liquid to give samples A, B, and C, respectively, and quantified by the method of the present invention, while quantified by liquid chromatography, and the results are compared and shown in the first cough. However, as shown in the table, there was good agreement between the two.

The buffer solution used was the same as in Example 6.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram of the structure of the microbial electrode used in the method of the present invention, in which 1 dissolved carbon dioxide electrode diaphragm, 2 microorganism layer, 3 carrier,
4 dialysis membranes, 5 PH electrodes, 6 internal liquids, and 7 microorganism electrodes are shown in their entirety. FIG. 2 shows one embodiment of a quantitative system set used in the method of the invention. In the figure, 7 microbial electrodes, 8, rubber backing, 9 flow cells, 10 N2 gas inlet, 11 buffer solution inlet,
12 sample injection ports, 13 PH meter or ion meter, 14 recorder. FIG. 3 is a graph showing the peak of electromotive force at the saturation level of the L-amino acid aqueous solution of Example 1 and its diluted solution, the vertical axis shows the electromotive force Mv of the electrode, and the horizontal axis shows time. FIG. 4 is a graph showing the relationship between the height MV of the peak in FIG. 3 and the concentration of L-amino acid solution in the flow cell.

Claims (1)

[Claims] 1. Between the diaphragm of the carbon dioxide gas electrode and the thin film with micropores covering it, the genus Escherichia, Citrobacter, Clostridium, Bacterium, Streptococcus, Pseudomonas, Bacillus, Myxococcus, and Lactobacillus are separated. The cells of a microorganism belonging to Bacteria belonging to the genus Aspergillus, Ascomycetes belonging to the genus Aspergillus, or yeast belonging to the genus Rhodotorula and having the decarboxylase activity of the L-amino acid to be quantified are treated with an organic solvent and/or a surfactant. The treated activated microbial cells are placed in a test solution containing the substrate L-amino acid under anaerobic conditions using an encapsulated microbial electrode or a microbial electrode in which a microbial membrane on which the microbial cells are immobilized is attached on a diaphragm of dissolved carbon dioxide. Immersion or contact, common logarithm value Log of the concentration C of the relevant L-amino acid
A method for quantifying L-amino acids using a microbial electrode, characterized in that the concentration of the relevant L-amino acid is determined using a comparative relationship between C and the electromotive force of the microbial electrode.