JPS591979B2 - Measuring method for glutamic acid and glutamine - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the production of L-glutamic acid and L-glutamic acid using microbial electrodes.
Concerning a method for measuring glutamine.

Conventionally, L-glutamate decarboxylase from pumpkin or Escherichia coli has been used as a method for quantifying L-glutamic acid.
An enzymatic method has been used in which the amount of CO2 gas generated by the reaction of aminobutyric EC02 with L-glutamic acid is measured by the Warburg method or the like using the formula (1) glutamate decarboxylase (1) using the formula 3.

Although the enzymatic method is accurate and excellent, it is difficult to use enzymes continuously, and expensive enzymes must be used anew for each measurement, making it uneconomical. Furthermore, autoanalyzers for colorimetric determination of generated CO2 gas are actually used, but they still suffer from the above-mentioned drawbacks and the equipment is complicated. A method of immobilizing the enzyme and using it continuously is also considered, but it has not yet been put to practical use.

Therefore, the present inventors conducted extensive research to develop a simple and accurate method for measuring glutamic acid, and as a result, we used a microbial electrode in which bacterial cells belonging to Escherichia coli were attached to a dissolved carbon dioxide gas electrode under anaerobic conditions. We were able to develop a new method for measuring glutamic acid and glutamine, which involves contacting the sample with the test solution. That is, the method of the present invention uses microbial cells to carry out the reaction of formula (1) under anaerobic conditions,
This method is characterized in that the generated CO2 gas is regulated using a carbon dioxide gas electrode. The present invention will be explained in detail below. The microorganism used in the present invention is Escherichia coli (
Escherichiacoli), glut
A strain with strong amatedecarboxylase activity is used, an example being Escherichia coli ATCC873.
9 etc. is suitable. Other bacteria such as Clostridium welchii can also be used. Bacterial cells can be prepared by culturing them in a conventional nutrient medium, and the obtained bacterial cells can be used as they are, or they can be lyophilized and used for a long period of time (one year or more), which is convenient.

The microbial electrode of the present invention is an ordinary dissolved carbon dioxide electrode (commercially available) with the above-mentioned microbial cells attached to the diaphragm, and the microbial cells are applied to a spacer such as a nylon net, a Miwapore filter, or a piece of paper. However, this can be easily produced by attaching a membrane such as a cellophane membrane having micropores that are impermeable to microorganisms to a diaphragm as shown in FIG.

Furthermore, microorganisms immobilized with collagen, acrylamide gel, etc. can also be used. 5 In Figure 1, 1 is the carbon dioxide gas electrode diaphragm (silicone membrane), 2 is the microorganism layer, 3 is the spacer, 4 is the dialysis membrane (cellophane membrane), 5 is the PH electrode, and 6 is the internal solution (NaHCO3- NaC
1 mixed liquid). When this microbial electrode is brought into contact with a test liquid such as a fermentation liquid under normal conditions, the microorganisms near the diaphragm assimilate or respire the nutrients (sugars, amino acids) in the test liquid, producing a large amount of carbon dioxide. It is difficult to measure glutamate because it releases The present inventors have found that when 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 activities of the 13 microorganisms mentioned above is completely suppressed, and therefore the reaction of equation (1) is suppressed. The present invention was completed based on the discovery that the process progresses quantitatively. The measurement system shown in FIG. 2 is one of the second embodiments of the present invention. In FIG. 2, 1 is a microbial electrode, 2 is a flow cell, 3 and 32 are rubber packings, 4 is an N2 gas inlet, and 5 is a carrier injection port. 6 is a sample injection port, 7 is a PH meter or ion meter, and 8 is a recorder.

The measuring method of the second invention will be explained below according to the system shown in FIG. First, flow the carrier liquid into the flow cell at a constant flow rate from 5 and while blowing N2 gas from 4, and record the electrode voltage output on recorder 8 (baseline). After the baseline is stabilized (within 1 hour) , 5 samples from 6 to 10 to 3 with a pulse time of 0.5 to 3 minutes.
This sample solution, which is sequentially injected at 0 minute intervals, is appropriately diluted with a carrier solution and reaches the flow cell.

In the flow cell, glutamic acid in the sample is decomposed by microbial enzymes according to the reaction of equation (1), and CO2 gas is generated. This CO2 gas passes through the diaphragm and P of the internal liquid.
Change H. The PH change is measured by a PH meter or ion meter 7 via a PH electrode, and recorded on a recorder 8. Logarithm of electrode output value and substrate concentration (】
0gC), a relationship as shown in FIG. 4 is observed, and Nernst's equation (2) holds true. (2) E in formula. is the asymmetric potential difference, R is the gas constant, T is the absolute temperature, and F is the Farade constant.

Using this equation (2), the substrate concentration of the test solution can be determined. Regarding the conditions at the time of measurement, the measurement ρH is 3.5
~5.5, the temperature is preferably in the range of 20 to 40°C, the contact time between the sample and the electrode is sufficient for 0.5 to 5 minutes, and the saturation value is usually reached in 3 minutes.

The measurement concentration range of the substrate is 10-1 to 10-4M, making it possible to measure a wide range. The carrier liquid used is an organic acid buffer such as citric acid, fumaric acid, or succinic acid, or a pyringine monohydrochloric acid buffer.

Especially contains NaCl and KH2PO2 (0.59/dl)
A pyridine monohydrochloride buffer is preferred.
In Figure 2, N2 gas is used to create an anaerobic condition, but it is not limited to N2 gas, as long as dissolved oxygen does not coexist, and replacement with other inert gases is also acceptable. Alternatively, a carrier containing no dissolved oxygen may be used.
When used under the above conditions, the activity will last for more than two weeks with continuous use. Since the method of the present invention uses microbial cells, it seems to be more susceptible to the effects of impurities such as amino acids and sugars than the enzymatic method, but when examining the substrate specificity, Table 1 shows that There is no substance other than glutamine that shows 570 or more for 100 glutamic acid.
As shown in Table 1, glutamine can also be measured in the same manner as glutamic acid using the method of the present invention.

This is because E. coli produces glutaminose (Glu).
This method is based on the fact that glutamic acid is produced from glutamine by the reaction of equation (3), and CO2 gas is produced by the reaction of equation (2). Other L-amino acids not listed in Table 1, i.e. Met, His
,Lys,PrO,Ser,Ileu,Phe,Leu
, Asp(NH2), Val, Thr, Ornl, malic acid, virupic acid, glucose, urea, etc. have no effect at all.

On the other hand, in the oxygen method (Warburg method) using freeze-dried Escherichia coli, Tyr is 45%, Arg is 80%, Try is 25%, and malic acid is 40%, relative to 100 glutamic acid, which is considerably affected. Pumpkin enzymes are greatly influenced by urea, making it impossible to measure glutamine or glutamine fermentation process using urea. The method of the present invention is also superior in terms of substrate specificity. As described above, the present invention provides a method for measuring glutamic acid and glutamate-6 which is simpler and more accurate than the enzymatic method.

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

After culturing for 20 hours, 50me of the culture solution was centrifuged to obtain wet bacterial cells.

This was thoroughly washed with 0.1M KC1 solution 50me and lyophilized to obtain 0.69 bacterial cells. Suspend (paste-like) 5q of freeze-dried bacterial cells in a small amount of water to a size of 10? A carbon dioxide gas electrode (Model E5O37, manufactured by Radiometer Co., Ltd.,
(Denmark) near the silicone diaphragm.

This microbial carbon dioxide electrode was inserted into a flow cell (capacity 0.5 mt) shown in FIG. 2 via rubber packing to assemble a measurement system as shown in FIG. PH4.4, 0.1 M pyridine monohydrochloric acid buffer (0.59/containing DlONaCl and KH2PO4) as carrier liquid.
was introduced at a flow rate of 5 to 5 in Figure 2, and N2 gas was blown in from 4 to 11/7 It'It through the flow cell, and the voltage output at this time was recorded on recorder 8. (Base line).

During the measurement, the temperature inside the flow cell was maintained at 3'C. After 1 hour, an aqueous solution of 465 glutamic acid and its diluted solution were sequentially injected from 6 to 6 at a rate of 1 d/melt with a pulse width of 3 minutes.

When this sample was diluted with a buffer and flowed into the flow cell, the microbial electrode gave an indication within a few seconds, and after 3 minutes, the indicated saturation level was reached, and a peak as shown in Figure 3 was recorded on the recorder. In Figure 3, the vertical axis shows the voltage output of the electrode (in m), and the horizontal axis shows time. A relationship is observed, with a slope of 60mV/PC
GH matched the coefficient of the Nernst term in equation (2) (theoretical value: 60.16 mV). - On the other hand, Previbacterium lactofermentum ATCCl3896 was added to the third
Aerated agitation culture was performed at 30°C using the medium shown in the table.

The resulting culture solution was centrifuged, and the supernatant was diluted 100 times to obtain sample A.

A fixed amount of glutamic acid as a reagent was added thereto to prepare samples B, C, and D. These samples were measured according to the system shown in Figure 2, and from the peak heights obtained (2
) The concentration of glutamic acid in each sample was determined according to the formula.

The results are shown in Table 4, and were in good agreement with the values measured by the conventional enzyme method (autoanalyzer method) for each sample. Example 2 Brevibacterium flavan AJ34O9 (FERM
-Pl684) was cultured with aeration and stirring at 30°C using the medium shown in Table 5.

After 50 hours of culture, centrifugation is performed, and the resulting supernatant is
It was diluted twice and measured according to the method of Example 1, and the substrate concentration of the supernatant was determined from equation (3) and was found to be 5.069/dl.

From this value, the amount of glutamine is determined by the amount of glutamic acid measured by an enzymatic method using pumpkin enzyme, which is 0.129/
The value obtained by subtracting dl is 4.949/91, and the value obtained by the conventional enzyme method (autoanalyzer method) using Escherichia coli and pumpkin enzymes is 4.939/
It was in good agreement with dl.

[Brief explanation of the drawing]

Fig. 1 is a schematic diagram of the microbial electrode of the present invention, Fig. 2 is a schematic diagram of the measurement system, Fig. 3 is a chart of voltage output of the microbial electrode, and Fig. 4 is a graph showing the relationship between substrate concentration and voltage output. .

Claims (1)

[Claims] 1. A microbial electrode with bacteria belonging to Escherichia coli attached near the diaphragm of the carbon dioxide electrode is brought into contact with the test solution under anaerobic conditions, and the difference between the logarithm of the substrate concentration (logC) and the voltage output of the microbial electrode is A method for measuring L-glutamic acid and L-glutamine, which comprises determining the substrate concentration using the proportional relationship.