JPS6042911B2 - Assay method for L-lysine - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for quantifying L-lysine using a microbial electrode.

Conventionally, the method for quantifying L-lysine has been to use L-lysine derived from Escherichia, coli or Bacterium chyadabellis.
Lysine decarboxylase (L-lysine decarboxylate)
ylase) to measure the amount of carbon dioxide gas generated by the reaction of the following formula (I) by manometry.
-Lysine decarboxylase.

+Co, (■) The Warburg pressure method determines the generated carbon dioxide, and the generated carbon dioxide is detected.

Both methods are accurate and excellent, but because it is difficult to use enzymes repeatedly, expensive enzymes must be used anew each time a measurement is performed, making them uneconomical. In some cases, the equipment is complex and expensive, and enzyme preparation is required. Although it is possible to immobilize the enzyme and use it continuously, this method has not yet been put to practical use.
On the other hand, as a chemical method for quantifying L-lysine, there is an acid ninhydrin method, but there are problems such as the need to take into account factors that interfere with the quantitative determination when certain amino acids coexist. Therefore, as a result of extensive research into a simple and accurate method for quantifying L-lysine, the present inventors found that
L-lysine belongs to microorganisms such as Escherichia, Bacterium, Streftococcus, Pseudomonas, Bacillus, Myxococcus, Clostridium, and Lactobacillus that belong to the Bacteria, and Aspergillus that belongs to the Ascomycetes. Microorganisms having decarboxylase activity or their freeze-dried cells are attached to a dissolved carbon dioxide electrode, and the microorganism electrode is immersed or brought into contact with a test solution containing L-lysine under anaerobic conditions, and the electromotive force is measured. When measuring, it was discovered that there is a proportional relationship between the electromotive force and the common logarithm value of the concentration of the substrate L-lysine,
The invention has been completed.

That is, the method of the present invention is characterized in that the reaction of formula (1) is carried out under anaerobic conditions using microbial cells, and the produced carbon dioxide is quantified using a dissolved carbon dioxide electrode. be.

The present invention will be explained in detail below. Any microorganism that can be used in the present invention can be used as long as it has a strong L-lysine decarboxylase activity; for example, among bacteria, Escherichia, Bacterium, Streptococcus, Pseudomonas, Bacillus, Myxococcus, The existence of L-lysine decarboxylase is widely known in various genera such as Clostridium and Lactobacillus, as well as in the genus Aspergillus among ascomycetes. Strains such as Escherichia coli ATCC23226
, Bacteriumc
adaveris) ATCC976 Streptococcus fae
calls) ATCCl2984, Pseudomonas Saccharophia (PseudOmOrlassaccha)
rOphia) ATCCl5946, Bacillus subtilis (Bacillus subtilis) ATCCl
5O37, Myxococcus virescens (MyxO
cOccus virescens) ATCC252O3
, Clostridium welsh (ClOstridi)
Lactobacillus casei
) ATCC7469, and Aspergillus niger (A
spergillus niger) ATCC6278,
etc. are preferably used, and the bacterial cells obtained by culturing each strain in a nutrient medium suitable for each strain under conditions suitable for each strain can be used as is, or it can be used after being freeze-dried. can.

Freeze-dried bacterial cells can maintain their activity for a long period of time (for example, one year or more) if stored in a cool place, so they can be conveniently used. These active microorganisms can also be immobilized with collagen, acrylamide gel, etc. using conventional techniques and used as immobilized microorganisms. However, even if an immobilized microbial membrane is not used, there is almost no difference in functions such as stability, so it is not necessary to use an immobilized microbial membrane, but its use is also included within the scope of the present invention. Of course it will be done. As shown in FIG. 1, the microbial electrode of the present invention is a conventional commercially available dissolved carbon dioxide electrode with the above-mentioned microbial cells attached to the membrane, and the cultured microbial cells are filtered or centrifuged. The separated and, if necessary, washed bacterial cells 2 are transferred to a carrier 3 such as nylon mesh, Millibore filter (1) Millibore trademark), Tomari paper, calcium carbonate, etc.
It can be easily produced by applying the membrane to a diaphragm, covering it with a membrane 4 such as a cellophane membrane having micropores that are impermeable to microorganisms, and attaching it to a diaphragm as shown in FIG.

As the thin film 4 having micropores that covers and supports the bacterial cell layer 2 of the microorganisms, any thin film 4 that does not pass through the bacterial cells of the microorganisms used in the present invention but allows carbon dioxide gas etc. to freely pass through may be used. 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 unnecessary. In Figure 1, 1 is the diaphragm (silicone membrane) of the dissolved carbon dioxide electrode, 2 is the microorganism layer, 3 is the carrier, 4 is the dialysis membrane (cellophane membrane), 5 is the PH electrode, and 6 is the internal solution (NaHCO-
7 shows 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 (
It is impossible to quantify L-lysine because it assimilates and/or respires (sugars, amino acids) and releases 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 respiration 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, the buffer solution is flowed into the flow cell 9 at a constant flow rate from 11 while blowing N2 gas from 10, the electromotive force of the electrode is recorded on the recorder 14, and the sample is injected from 12 for 0.5 to 5 minutes. This sample solution is injected sequentially at intervals of 1 to 3 samples, and this sample solution is appropriately diluted with a buffer solution and reaches the flow cell 9.

In the flow cell 9, L-lysine 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 the internal liquid P
Change H. The PH change is measured by a PH meter or ion meter 13 via a PH electrode 5, and a recorder 1
Recorded in 4. A relationship as shown in FIG. 4 is observed between the electromotive force E of the electrode and the common logarithm 10 gC of the concentration C of the substrate L-lysine, 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 measurement is 3.5 to 6.5.
The s temperature is preferably in the range of 20 to 40°C, and the contact time between the sample and the electrode is sufficient for 0.5 to 5 minutes, and the saturation value is generally reached in 3 minutes.

The measurement concentration range of the substrate is 10-1 to 10-4M, allowing measurement over a wide range, and the linearity of E<510gC is very good. The buffer used is an organic acid buffer such as citric acid, fumaric acid, or succinic acid, or a pyridine-hydrochloric acid buffer.

Especially NaCl and KH2PO4 (0.5y/dl each)
), and pyridoxal-5″-phosphoric acid (a).1v/
A pyridine-hydrochloric acid buffer containing e) 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. It is also possible to use a carrier that does not contain dissolved oxygen.

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 active microbial cells, it is thought to be susceptible to impurities such as amino acids other than L-lysine, organic acids, and sugars. As shown in the table, L-
No effect of 10% or more on 100% lysine was observed.

In addition, other amino acids not shown in Table 1, L-serine, L
-Threonine, L-cysteine, L-cystine, L-methionine, L-asparagine, L-glutamine, L-phenylalanine, L-aspartic acid, L-histidine, L-arginine, L-ornithine hydrochloride, L-citrulline, No effect was observed with L-isoleucine, DL monolactic acid, tartaric acid, α-ketoglutaric acid, succinic acid, malic acid, fumaric acid, citric acid, glucose, urea, etc.

This result shows that L-cysteine is 8% and L-cysteine is 8% relative to 100% L-lysine, which is the value of the acidic ninhydrin method under conditions (475 mμ) that improves selectivity to L-lysine the most.
- Cystine 3%, L-tyrosine 11%, L-histidine 4%, L-proline 17%, L-ornithine hydrochloride 8%
Compared to this selectivity, the method of the present invention can be said to have extremely excellent selectivity. As mentioned above, the method of the present invention is similar to other methods, namely, the Warburg pressure method,
We believe that this method provides a simpler and more accurate method for quantifying L-lysine than the autoanalyzer method using an enzyme or the acidic ninhydrin method.

Example 1 Escherichia colli (EscherihiacOll)
ATCC23226 was cultured in a shaking flask at 30°C using the medium shown in Table 2.

After incubation for 2 hours, 50 ml of the culture solution was centrifuged, and the 0.0 ml of culture solution was centrifuged.
1M. ．． The cells were thoroughly washed with 50 mt of KC1 solution and freeze-dried to obtain 0.6V bacterial cells.

Dissolve 1 to 2 m9 of freeze-dried bacterial cells in a small amount of water to make a paste 2, and apply it to a nylon mesh 3 with a diameter of 1-mm, and use a cellophane membrane 4 to connect it to a dissolved carbon dioxide electrode as shown in Figure 1. (E5O3, Radiometer, Denmark) was mounted on a silicone film 1.

Using this microbial electrode, it was inserted into a flow cell (capacity: 0.5 ml) shown in FIG. 2 via rubber backings 8, 8'', and a measurement system as shown in FIG. 2 was assembled.

The buffer solution has a pH of 5. 0.1M pyridine-hydrochloric acid buffer solution (a). 5y/Dt(7)NaCl
and KH2PO4, 0.1y/'pidoxalu5''
(containing phosphoric acid) flows from 11 in Fig. 2 at a flow rate of 4 m1/min, and from 10 blows N2 gas at a flow rate of 0.2 e/min to pass through the flow cell 9, and the electrode 7 measures the electromotive force. Therefore, it was connected to a PH meter 13 and a recorder 14.

During the measurement, the temperature inside the flow cell was maintained at 30°C. As a sample, a 5y/e aqueous L-lysine solution and its diluted solution were sequentially injected from the sample injection port 12 at a rate of 1 ml/min for 3 minutes. At the same time as this sample was diluted with a buffer solution 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.

In FIG. 3, the vertical axis represents the electromotive force m of the electrode, and the horizontal axis represents time. The relationship shown in Figure 4 is seen between the height of each peak in Figure 3 and the L-lysine concentration in the flow cell 9, and the slope is 5.
8rr1V/PCLy8 almost matched the coefficient of the Nernst term in equation (■). (Theoretical value: 60.16n
1V, 30°C) On the other hand, Brevibacterium Lactofermentum ATCCl3869 was cultured with aeration at 30°C using the medium shown in Table 3.

Dilute the culture solution 2@ to make sample A, and add L of reagent to this.
- Samples B, C, and D were prepared by adding a known amount of lysine.

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

The results are shown in Table 4, and the values for each sample were in good agreement with the values measured by the conventional Warburg manometry method using an enzyme derived from Bacterium cyadaveris.

Example 2 Bacterium
adaveris) ATCC976O was cultured in a shaking flask at 30°C using the medium shown in Table 5.

50 mt of the obtained culture solution was collected, and 0.1M. ．． KC1
After washing twice with the solution, drying in a vacuum desiccator to a 0.
3y of dried bacterial cells were obtained, and a microbial electrode was constructed.

For time-lapse samples A, B, C, and D of L-lysine fermentation liquid obtained in the same manner as in Example 1, the concentration of L-lysine was determined by this method and the conventional acid ninhydrin method. Six tables were obtained. The analytical values obtained by both methods for each sample were in good agreement.

Example 3 Clostridium Welsh (ClOstridium)
mwelchll) ATCCl3l24 with 3% tryptic digest, 2% glucose, 100 pq/l
A flask culture was carried out using a culture solution containing pyridoxal, 0.5% L-lysine hydrochloride, and meat pieces added thereto at 30° C. and 2° C. with hydrogen gas.

A microbial electrode was constructed using 50 mL of the culture solution according to the method of Example 2, and the L-lysine concentration was determined for the L-lysine fermentation solution of the same sample as in Example 2. The value was in good agreement with the value obtained by the method. Example 4 Lactobacillus casei (LlctObacillus casei)
●Casei) ATCC7469 (NO.l) 10%
Skimmed milk powder, 0.5% L-lysine hydrochloride, 100μy/e
Streptococcus faecalis (Streptococcus faecalis) with a culture solution consisting of pyridoxal.
aIis) ATCCl2984 (NO.2), Pseudomonas satsukalovia (PseudOmOnassa)
ccharOphia) ATCCl5946 (NO.3
), Bacillus subtilis (Bacillus subtilis)
tllls) ATCCl5O37 (NO.4), Myxococcus virescens (MyxOcOccusvi
Rescens) ATCC252O3 (NO.5), 1% meat scratches, 1% polypeptone, NaClO. 5%,
Aspergillus niger (A
spergilusniger) ATCC6278 (
NO. 6) with 2% glucose, 0.1% KH2PO4,
Each flask was shaken and cultured in a medium of Koji juice supplemented with lime carbonate, and a microorganism electrode was constructed by the method of Example 2.
Regarding L-lysine fermentation liquids A, B, and C of samples similar to L
- When the lysine concentration was determined, as shown in Table 8, it agreed well with the value determined by the conventional ninhydrin method.

[Brief explanation of the drawing]

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 membrane, 5 PH electrode, 6 internal solution, 7 shows the entire microorganism electrode. 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 port, 13 PH meter or ion meter, 14 Recorder Figure 3 is a graph showing the peak of the electromotive force at the saturation level of the L-lysine aqueous solution of Example 1 and its diluted solution, and the vertical axis is the horizontal electromotive force Mvl of the electrode. The 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-lysine solution in the flow cell.

Claims (1)

[Claims] 1. A microbial electrode in which microbial cells having L-lysine decarboxylase activity are encapsulated, or a microbial membrane in which the microbial cells are immobilized, is placed between the diaphragm of the carbon dioxide gas electrode and a thin film having micropores covering the membrane. A microbial electrode attached to a diaphragm of dissolved carbon dioxide is immersed or brought into contact with a test solution containing L-lysine under anaerobic conditions, and the common logarithm LogC of the L-lysine concentration C and the origin of the microbial electrode are determined. A method for quantifying L-lysine using a microbial electrode, characterized in that the concentration of L-lysine is determined using a proportional relationship with electric power.