JPH06169798A - Device for measuring lactic acid and method using the same - Google Patents

Abstract

PURPOSE:To provide the device and method for measuring the amounts of D-lactic acid and L-lactic acid in a short time in good accuracy and in simple operations. CONSTITUTION:The flow style device for measuring the D-lactic acid and L- lactic acid is provided with a mechanism (24) for injecting a specimen into a flowing carrier from its upstream, a reactor (25) therein having immobilized enzymes containing D-lactic acid dehydrogenase and L-lactic acid dehydrogenase, a L-lactic acid oxidase-immobilized product (28), and an electrode (29) for detecting an electrode-active substance increasing or decreasing when the L- lactic acid in the specimen is oxidized with the L-lactic acid oxidase.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a rapid and simple measuring apparatus and method for lactic acid using immobilized enzyme.

[0002]

2. Description of the Related Art Lactic acid fermentation has been widely used in the field of food industry. For example, lactic acid fermentation by microorganisms not only produces lactic acid and milk drinks, but also cheese, butter, sake,
It is used to produce soy sauce, miso and pickles. Microorganisms used for these lactic acid fermentations include lactic acid bacteria and filamentous fungi. Among these, lactic acid bacteria produce only D-lactic acid, and produce only L-lactic acid, depending on the bacterial species.
Those which produce a mixture of D-lactic acid and L-lactic acid are known, and the measurement of D-lactic acid and L-lactic acid in the fermentation process is essential for fermentation control, discovery of contamination, and the like.

Conventionally, the measurement of D-lactic acid was carried out by using D-lactate dehydrogenase (hereinafter abbreviated as D-LDH) and the coenzyme nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD) to produce reduced nicotinamide. Adenine dinucleotide (hereinafter abbreviated as NADH) or pyruvic acid was measured by absorptiometry and fluorescence intensity method. However, this measuring method has various problems depending on the properties of D-LDH.

First, since the reaction efficiency is low, the amount of enzyme required is too large at a practical level, resulting in a high analysis cost. In order to reduce the amount of enzyme to be used, it is conceivable to lengthen the reaction time or adjust the reaction temperature, but it has been a negative factor such as more complicated operation and extension of the measurement time. Secondly, the equilibrium of the reaction involving D-LDH is inclined toward the production of D-lactic acid from NADH and pyruvic acid, so that the reaction is stopped before all D-lactic acid is completely changed. . Therefore, means such as consumption of the produced pyruvate by glutamate pyruvate transaminase is required, which complicates the reaction process and raises the analysis cost.

When not only D-lactic acid but also L-lactic acid is measured, first, D-lactic acid is analyzed, and then the same measurement as D-lactic acid is carried out. LDH). Therefore, the operation becomes complicated, and the measurement time and cost are doubled. Further, in the measuring method using only lactate dehydrogenase, the general detection means of the reaction is the change in absorbance in the ultraviolet region of NAD or NADH which changes depending on the reaction, and there is a drawback that the sample is easily affected by turbidity and coloring substances. there were.

[0006]

As described above, the conventionally disclosed measuring methods for D-lactic acid and L-lactic acid cannot be said to be sufficiently practical measuring methods. The present invention relates to D-LDH and L
An object of the present invention is to provide a measuring device and a measuring method that can measure two components of D-lactic acid and L-lactic acid accurately and easily in a short time using an immobilized body of LDH.

[0007]

The gist of the present invention is illustrated by the following embodiments, but the invention is not limited thereto.

(1) Enzyme-immobilized body containing D-lactate dehydrogenase and L-lactate dehydrogenase, a mechanism for bringing nicotinamide adenine dinucleotide and a sample into contact with the enzyme-immobilized body, and a mechanism for measuring L-lactic acid An apparatus for measuring D-lactic acid and L-lactic acid, which comprises:

(2) A mechanism for injecting a sample into a carrier to be fed from upstream, a reactor containing an enzyme immobilization body containing D-lactate dehydrogenase and L-lactate dehydrogenase, and L-lactic acid. Flow-type D-lactic acid and L-lactic acid, which are equipped with an oxidase-immobilized body and an electrode for detecting an electrode active substance that increases or decreases when L-lactic acid in a sample is oxidized by L-lactic acid oxidase measuring device.

(3) In the state where the immobilized enzyme containing D-lactate dehydrogenase and L-lactate dehydrogenase and the carrier containing the sample are in contact with each other, it has a mechanism of stopping the liquid feeding of the carrier or lowering the flow rate. (2) The measuring device as described above.

(4) On the upstream side of the reactor, an electrode active substance that increases or decreases when L-lactic acid in a sample is further oxidized by the L-lactate oxidase-immobilized body and L-lactate oxidase is detected. The measuring device according to (2), which is provided with an electrode for

(5) a. The sample and nicotinamide adenine dinucleotide are brought into contact with an enzyme-immobilized body containing D-lactate dehydrogenase and L-lactate dehydrogenase to carry out a conversion reaction between D-lactic acid and L-lactic acid in the sample, and then L -A step of measuring lactic acid, b. A method for measuring D-lactic acid and L-lactic acid, which comprises the step of measuring L-lactic acid in a sample.

(6) a. A step of bringing a sample and nicotinamide adenine dinucleotide into contact with an enzyme-immobilized body containing D-lactate dehydrogenase and L-lactate dehydrogenase to carry out a conversion reaction between D-lactic acid and L-lactic acid in the sample; and b. A method for measuring D-lactic acid and L-lactic acid, which comprises a step of electrochemically detecting an electrode active substance that increases or decreases when L-lactic acid in a sample is oxidized by L-lactic acid oxidase. In step b, using L-lactic acid standard solution as a sample,
-The calibration curve 1 showing the relationship between the lactic acid concentration and the output value is obtained.-Using the L-lactic acid standard solution as a sample, the step a is performed, and then the step b is performed on the sample to show the relationship between the L-lactic acid concentration and the output value. The calibration curve 2 shown below is obtained, and step a is performed using a D-lactic acid standard solution as a sample, and then step b is performed for the sample to obtain a calibration curve 3 showing the relationship between the D-lactic acid concentration and the output value. In step b, a measurement sample is used as a sample, and an output value of 1 is measured, and when a measurement sample is used as a sample, step a is performed, and then step b is performed, an output value 2 is measured, and an output value 1, an output value 2 and a calibration curve are obtained. 1. Calculate the amount of D-lactic acid and the amount of L-lactic acid in the measurement sample from the calibration curve 2 and the calibration curve D-
Method for measuring lactic acid and L-lactic acid.

(7) A flow type equipped with a mechanism for sending a buffer solution, an L-lactate oxidase-immobilized body, and an electrode for detecting an electrode active substance that increases or decreases when L-lactic acid is oxidized. A measuring device, wherein an enzyme-immobilized body having D-lactate dehydrogenase and L-lactate dehydrogenase immobilized thereon is arranged in front of the L-lactate oxidase immobilized body, and the D-lactate dehydrogenase and L-lactate dehydrogenase are arranged.
-A measuring device having a mechanism for bringing nicotinamide adenine dinucleotide and a sample into contact with an enzyme-immobilized body in which lactate dehydrogenase is immobilized.

(8) The L-lactate oxidase-immobilized product is L-
The measuring device according to (7), which is a column containing a carrier on which lactate oxidase is immobilized, or a column of immobilized enzyme, which is attached to an electrode.

(9) The measuring apparatus according to (7), wherein the enzyme-immobilized body in which D-lactate dehydrogenase and L-lactate dehydrogenase are immobilized has both enzymes immobilized on a carrier and packed in a column.

(10) With the nicotinamide adenine dinucleotide and the sample in contact with the D-lactate dehydrogenase and L-lactate dehydrogenase-immobilized body, the solution transfer is stopped for a certain period of time or for a certain period of time. The measuring device according to (7), which is provided with a mechanism for reducing the flow rate of the liquid.

(11) The flow path system having the L-lactate oxidase-immobilized body and the electrode has two flow paths in parallel, and the D-lactate dehydrogenase and the L-form are added in front of the L-lactate oxidase-immobilized body on one side. -The measuring apparatus according to (7), in which an enzyme-immobilized body on which lactate dehydrogenase is immobilized is arranged.

(12) When the sample is brought into contact with the immobilized D-lactate dehydrogenase or L-lactate dehydrogenase using the measuring device described in (7), the presence and the presence of nicotinamide adenine dinucleotide Measurement method to measure twice if not. In addition, when a sufficient time is taken for the conversion reaction of D-lactic acid and L-lactic acid, the sample becomes a mixture of about 1: 1,
In (6), since the calibration curve 2 and the calibration curve 3 substantially match,
Even two calibration curves can be calculated. And (6) should be interpreted as a generalized one including this case.

[0020]

The reaction of D-LDH and L-LDH is shown in the following formula. D-LDH reaction: D-lactic acid + NAD → pyruvic acid + N
ADH L-LDH reaction: L-lactic acid + NAD → pyruvic acid + N
ADH Both reactions are reversible, but the equilibrium of both enzymes is inclined toward the formation of lactic acid and NAD. for that reason,
When a large amount of D-LDH is contacted with a sample in the presence of NAD, it hardly changes to pyruvic acid. The same applies to L-LDH.

However, the present inventors found that the sample and NAD were D-
Upon contact with the LDH and L-LDH mixture, L in the sample
It was found that -lactic acid is converted to D-lactic acid, and D-lactic acid is converted to L-lactic acid. It is considered that this phenomenon is that lactic acid was once oxidized to pyruvic acid, and lactic acid was regenerated by the catalytic action of D-LDH and L-LDH existing in the vicinity of the generated pyruvic acid and NADH.

As a more interesting fact, it was found that even in the mixed sample of D-lactic acid and L-lactic acid, the conversion rate of each optical isomer of lactic acid was independent. That is, D-
A sample and NAD are brought into contact with a proximity immobilization body of LDH and L-LDH, and the amount of L-lactic acid after contact with the immobilization body is detected. For example, the object of the present invention can be achieved by detecting with an immobilized L-lactate oxidase and a hydrogen peroxide electrode. When a sample containing only various concentrations of L-lactic acid was brought into contact, a concentration range where a linear relationship was established between L-lactic acid and the electrode output value was observed. Therefore, by selecting one point in this concentration region and adding various concentrations of D-lactic acid to the L-lactic acid solution, another calibration curve can be obtained. Let this calibration curve be L1. Next, let L2 be the calibration curve obtained from the relationship between the sample containing only various concentrations of D-lactic acid and the current output value. Then, the slopes of L1 and L2 coincide, and the intercept of L1 coincides with the current output value when L-lactic acid at the relevant concentration is brought into contact. In this system, the same result can be obtained by replacing L-lactic acid and D-lactic acid.

This means that the relationship between the concentrations of D-lactic acid and L-lactic acid and the current output value is linear and independent, and even if D-lactic acid and L-lactic acid are mixed in the actual sample, If the contained L-lactic acid concentration is determined separately,
It shows that the amount of D-lactic acid can be calculated. As a matter of course, D-LDH, L-LDH immobilized body, sample and NA
The longer the contact time of D, the higher the conversion rate of D-lactic acid and L-lactic acid. If the reaction is carried out for a sufficient time, finally D-lactic acid and L-lactic acid are equilibrated and a racemate is completed.
However, the method of obtaining the D-lactic acid amount and the L-lactic acid amount by equilibrating requires a long time for the reaction when D-LDH and L-LDH are immobilized, and requires continuous measurement. Is inappropriate. In a general enzymatic reaction, a linear relationship is established between the reaction time and the conversion rate when the conversion rate of the substrate is about 20% or less. Therefore, in the case where the amount of enzyme used for immobilization is 20% or less, the sensitivity increases almost linearly as the contact time increases. However, since the reaction rate gradually decreases thereafter, even if the flow rate of the liquid feed is slowed and the contact time is lengthened, the effect obtained thereby becomes low. Even if the liquid feeding is stopped or the flow rate is decreased, the conversion rate can be practically measured in a short time if the conversion rate is about 80%.

The mode of stopping the liquid feeding or reducing the flow velocity of the liquid feeding will be described later. D-LDH, LL
In order to extend the contact time between the DH-immobilized body and the sample and NAD, the flow rate of the solution may be reduced while the D-LDH and L-LDH-immobilized body is in contact with the sample and NAD. Further, if the sample is brought into contact with the D-LDH and L-LDH-immobilized body and the liquid feeding is stopped and the reaction is carried out for a long time, a conversion rate of 100% is possible. In this case, lactic acid is D-lactic acid: L-lactic acid = 1:
A racemization of 1 is produced.

In the present invention, the D-LDH- and L-LDH-immobilized products are used to carry out the measurement under a constant reaction condition, so that accurate measurement can be performed even if the conversion rate is not 100%.
Specifically, the following measuring devices can be exemplified. The first device divides the sample into two, one of which is contacted only with L-lactate oxidase and used for L-lactic acid measurement. The other is D-LD
The H, L-LDH and NAD are contacted for a certain time. Then, it is brought into contact with L-lactate oxidase and used for the measurement of L-lactic acid after the conversion reaction. As shown in FIG. 1, it is possible to measure easily and quickly by dividing the flow path into two directions.
Alternatively, the sample flow may be measured twice by two measurement systems without branching.

The second device is the L-lactate oxidase-immobilized column (44), the hydrogen peroxide electrode (45), and the D-
LDH and L-LDH immobilized column (46), L-
This is a device in which a lactate oxidase-immobilized column (47) and a hydrogen peroxide electrode (48) are connected in series (Fig. 3). First,
The sample is contacted with an L-lactate oxidase-immobilized column (44) to detect L-lactic acid, followed by D-LDH and L-LDH.
And NAD for a certain period of time, and then contacted with an L-lactate oxidase-immobilized column (47) to determine the detected value of L-lactic acid. The third device is a flow-type measuring device (FIG. 2) that has an immobilized column (25) for D-LDH and L-LDH, and is equipped with an L-lactic acid detection mechanism on the downstream side thereof. When the sample is contacted with D-LDH and L-LDH, the case where NAD is present and the case where NAD is not present are measured, and the detection value of L-lactic acid in both is obtained. At this time, a sample in which NAD is added to the sample and a sample in which NAD is not added may be measured, or a method of using two types of buffer solutions, that is, a case of measuring with a buffer solution containing NAD and a buffer solution not containing NAD may be used. Regarding this aspect, Example 2
Described in.

For the actual measurement, first, a standard curve is prepared from the current values obtained by measuring the standard solutions of D-lactic acid and L-lactic acid. Calibration curve 1 is a calibration curve for L-lactic acid and is measured when the conversion reaction of D-lactic acid and L-lactic acid is not performed. That is, without contacting the D-LDH or L-LDH-immobilized body, or before contacting only with the L-lactate oxidase-immobilized body, or when NAD is contacted with D-LDH, L-
-This is the case where the sample was measured without contact with the LDH-immobilized body. Under these conditions, no current value can be obtained even if D-lactic acid is injected.

Calibration curve 2 shows that L-lactic acid was D in the presence of NAD.
It is a calibration curve obtained from the current value after passing through the -LDH and L-LDH immobilized bodies. Calibration curve 2 is L compared to calibration curve 1
-The proportion of hydrogen peroxide produced from lactic acid is low. The calibration curve 3 is created from the current value obtained from the standard solution of D-lactic acid under the same conditions as the calibration curve 2. In the case of measuring an actual sample, a current value 1 obtained in the same step as the calibration curve 1, a calibration curve 2 and a current value 2 obtained in the same step as the calibration curve 3 are obtained.

The amounts of D-lactic acid and L-lactic acid are calculated by the methods exemplified below. The L-lactic acid amount is calculated by substituting the current value 1 into the calibration curve 1. The calculated L-lactic acid concentration is substituted into the calibration curve 2 to calculate the current value of L-lactic acid contributing to the current value 2. L− that contributes to the current value 2 from the current value 2
Subtracting the lactic acid current value gives the D-lactic acid current value. The amount of D-lactic acid can be calculated by substituting the current value of this D-lactic acid contribution into the calibration curve 3.

The present invention is characterized in that D-LDH and L-LDH are immobilized and used. When immobilized and used, there are advantages that the enzyme can be repeatedly used and that the reaction conditions of the enzyme can be easily adjusted. The method for immobilizing the enzyme is not particularly limited, and an adsorption method, a chemical bonding method, an encapsulation method or the like can be used. Above all, a chemical bonding method that can form a strong immobilized body is preferable. As the carrier used for immobilization, diatomaceous earth, silica gel, glass beads, alumina, ceramics, carbon, activated carbon, molecular sieve, silicone rubber, cellulose, agarose, amino acid-based polymer and the like can be used. Examples of the chemical bonding method include a method in which an amino group is introduced on the surface of a carrier with an aminosilanization reagent, formylation is further performed using a polyfunctional aldehyde such as glutaraldehyde, and then an enzyme is contacted to immobilize it. . The form of the immobilized enzyme may be a method of immobilizing it on a membrane on the surface of an electrode, a method of immobilizing it on a carrier and filling it in a reactor such as a column, a reactor using a membrane or a hollow fiber, and the like. Among them, the method of filling the column with the immobilized carrier is advantageous because the contact time between the sample and the immobilized enzyme can be lengthened and the conversion rate can be increased. A column with a large volume is advantageous because it can be packed with more carriers and the reaction time becomes longer. However, if the reaction time is long, the measurement time becomes long and it is disadvantageous for the measurement.
It is about 100 μl. The material of the column is acrylic,
A fluororesin, a vinyl chloride resin, a metal such as glass or stainless steel, or a combination thereof can be used.

The mixing ratio of D-LDH and L-LDH that acts to cause the conversion reaction of D-lactic acid and L-lactic acid is not particularly limited, but the ratio of L-LDH to D-LDH is expressed as activity. In this case, it can be used from about 1/10 to about 20 times. 1 / 2-10 in this concentration range
The range is preferably double, and more preferably 1 / 2-5. The LDH activity is generally defined by measuring the rate of producing lactic acid from pyruvic acid.

The conversion reaction of lactic acid in the sample is as described in D above.
-Because of the action of LDH reaction and L-LDH reaction, D-
LDH and L-LDH are preferably fixed in close proximity so that the sample and NAD are in contact with each other almost simultaneously. Specifically, D
A method of stacking the LDH-immobilized membrane and the L-LDH-immobilized membrane,
There are a method of mixing a carrier on which D-LDH is immobilized and a carrier on which L-LDH is immobilized, a method of mixing a D-LDH solution and an L-LDH solution, and then immobilizing on a carrier or a membrane. However, since D-LDH and L-LDH generally have low enzymatic activity and slow enzymatic reaction, the method of immobilizing them on the electrode surface cannot always sufficiently perform the conversion reaction of lactic acid.
It is desirable to use it after immobilizing it on a carrier and filling it in a reactor such as a column.

The NAD may be added to the sample or the carrier. When NAD is added to the sample, it is preferably about 2 mM to 20 mM,
This method is suitable when the number of samples is small. Also, when adding to the carrier by the flow method, always use D-LD.
Immobilized H, L-LDH and NAD are in contact with each other, and the use effect is higher than when added to the sample.
About M to 10 mM is preferable. This method is suitable when the number of samples is large.

Of course, if the contact time between the D-LDH / L-LDH-immobilized product and the sample / NAD is extended, the NAD may be small. In addition, as described above, since the reaction rate of the D-LDH and L-LDH-immobilized body is relatively slow, in order to obtain sufficient sensitivity or to increase the conversion rate to approach the equilibrium state,
A mechanism may be provided in which, while the D-LDH or L-LDH-immobilized body is in contact with NAD and the sample, the solution feeding is stopped for a certain period of time or the flow rate of the liquid feeding is reduced for a certain period of time.

When the liquid feeding is stopped for a certain period of time, the liquid feeding is preferably started at the time when the whole sample enters the D-LDH- and L-LDH-immobilized body. In the range which does not impair the quickness of measurement, which is a characteristic of the above, it is practically preferable to set the time within 5 minutes in order to measure in a short time. Further, when the flow rate of the liquid transfer is reduced for a certain period of time, at least the beginning of the sample part is D-LD.
It is necessary to reduce the flow rate before leaving the H, L-LDH-immobilized body, and preferably the head of the sample part is D-LDH, L.
-Reduce the flow rate when entering the LDH-immobilized body. The time for maintaining the low speed is until the whole sample has passed through the D-LDH- and L-LDH-immobilized body, and similarly, it is preferably within 5 minutes for measuring in a short time.

As a means for stopping the liquid feeding of the sample solution in the D-LDH or L-LDH-immobilized body for a certain period of time or reducing the flow rate of the liquid feeding for a certain period of time, for example, from the sample injecting section to D-LDH
-Calculate from the length of the pipe to the LDH / L-LDH immobilized body, the pipe diameter, and the pumping speed of the pump at that time, or measure it in advance and measure the injection time from the D-L
It is advisable to determine the time required to reach the DH / L-LDH-immobilized body and change the control value of the pump flow rate when the time reaches the D-LDH / L-LDH-immobilized body.

These specific methods include, for example, using a specific electronic circuit or computer,
A method is used in which the flow rate of the pump is controlled via the D / A converter and the arrival time from the sample injection to the D-LDH / L-LDH immobilized body is accurately monitored. Further, as described in the second embodiment, the loop portion connected to the switching valve has a D-
It is also possible to increase the conversion rate by connecting the LDH and L-LDH immobilized body reactors and retaining the sample in the reactor.

Next, a method for selectively measuring L-lactic acid will be described. L-lactic acid must be measured by a method that does not affect the presence of substances other than L-lactic acid such as D-lactic acid and NAD. And the measuring method using L-lactate oxidase can be used. The reaction of L-lactate oxidase is shown in the following formula. L-lactic acid + O ₂ → pyruvic acid + H ₂ O ₂ This reaction is not affected even when D-lactic acid and NAD coexist, and is therefore suitable for accurately quantifying only L-lactic acid.

L-lactic acid can be quantified using L-lactate oxidase by detecting decreased oxygen or increased hydrogen peroxide. Further, the measurement can be performed by interposing an electron carrier such as dichloroindophenol, potassium ferricyanide, benzoquinone, a so-called mediator, or the like.
Electrochemical measurement method that measures increase and decrease of electrode active substances such as oxygen, hydrogen peroxide, etc. by current value by electrodes is not affected by turbidity of sample and coloring substances compared with measurement using spectrophotometer It is preferable because it is easy to operate.

Although L-lactate oxidase can be used in a solution, it has advantages that it can be used repeatedly after being immobilized and that the reaction conditions of the enzyme can be easily adjusted. As the immobilization method, the same adsorption method, chemical bonding method, encapsulation method and the like as described above for D-LDH and L-LDH can be used, and they can be used by being contained in a reactor such as a column. Further, a membrane in which L-lactate oxidase is fixed with a cross-linking agent such as glutaraldehyde, formaldehyde, succinyl aldehyde can be attached to the electrode and used. Albumin, globulin,
Other proteins such as gelatin can be added to crosslink the L-lactate oxidase.

As the oxygen electrode for measuring the consumed oxygen, various known ones such as a carbani type and a Clark type can be used.
As the hydrogen peroxide electrode for measuring the hydrogen peroxide produced, a known one using carbon, platinum, nickel, palladium or the like for the anode substrate and silver or the like for the cathode side can be used. Generally, platinum is often used as the anode because of its low overvoltage against hydrogen peroxide and high sensitivity. And a polysiloxane film on the electrode surface,
An electrode having a permselective membrane such as an acrylic resin membrane, a protein membrane and an acetylcellulose membrane is preferable from the viewpoint of removing obstacles.

The electrode system is composed of a working electrode and a counter electrode 2
A hydrogen peroxide electrode or an oxygen electrode can be used as the electrode. From the viewpoint of stability and accuracy, a three-electrode structure including a working electrode, a reference electrode and a counter electrode is preferable. As a carrier to be sent, a buffer solution or the like which has a buffering capacity at around pH 7 which is a suitable pH for D-LDH, L-LDH and L-lactate oxidase and which does not exert an electrochemical influence on the electrode is used. To be

[0043]

The contents of the present invention will be described in more detail with reference to the following examples, but of course the present invention is not limited thereto.

Example 1 (1) Production of L-lactate oxidase-immobilized column 150 mg of fire-resistant brick (30 to 60 mesh), which was calcined diatomaceous earth, was well dried and anhydrous 10% γ-aminopropyltriethoxysilane. After soaking in a toluene solution for 1 hour, it is thoroughly washed with toluene and dried. After the aminosilane-treated carrier was immersed in 5% glutaraldehyde for 1 hour, it was washed thoroughly with distilled water and finally pH adjusted.
Replace with 7.0, 100 mM sodium phosphate buffer and remove this buffer as much as possible. This formylated refractory brick was brought into contact with 200 μl of a solution prepared by dissolving L-lactate oxidase (manufactured by Sigma) at a concentration of 50 units / ml in 100 mM sodium phosphate buffer having a pH of 7.0, and at 0 to 4 ° C. for 1 day. Leave and fix. The enzyme-immobilized carrier was packed in an acrylic column having an inner diameter of 3.5 mm and a length of 30 mm to give an L-lactate oxidase-immobilized column.

(2) Manufacture of hydrogen peroxide electrode A side surface of a platinum wire having a diameter of 2 mm is coated with heat-shrinkable Teflon,
One end of the wire is smoothed with a file and a No. 1500 emery paper. Using this platinum wire as a working electrode, a 1 cm square platinum plate as a counter electrode, and a saturated calomel electrode as a reference electrode, 0.1M
Electrolyte in sulfuric acid at +2.0 V for 10 minutes. After that, the platinum wire was washed well with water and then dried at 40 ° C for 10 minutes.
It is immersed in an anhydrous toluene solution of 10% γ-aminopropyltriethoxysilane for 1 hour and then washed. 20 mg of bovine serum albumin (Fraction V, manufactured by Sigma) is dissolved in 1 ml of distilled water, and glutaraldehyde is added to the solution to make the concentration 0.2%. 5 μl of this mixed solution was quickly put on a platinum wire prepared in advance, and dried and cured at 40 ° C. for 15 minutes to form a hydrogen peroxide selective permeable membrane, which was used as a hydrogen peroxide electrode. An Ag / AgCl reference electrode was used as the reference electrode, and a conductive pipe was used as the counter electrode.

(3) Production of D-LDH and L-LDH-immobilized column pH was 7.0 and 100 m on calcined diatomaceous earth (refractory brick) formylated in the same manner as in the L-lactate oxidase immobilization method.
500 μ solution of M sodium phosphate buffer mixed with 300 units / ml of D-LDH (manufactured by Boehringer Yamanouchi) and 900 units / ml of L-LDH (manufactured by Sigma)
1 is contacted and left at 0 to 4 ° C. for 1 day for immobilization. This enzyme-immobilized carrier was packed in an acrylic column having an inner diameter of 3.5 mm and a length of 30 mm to prepare a column on which D-LDH and L-LDH were immobilized.

(4) Measuring device L-lactic acid and D-lactic acid are measured by the flow type measuring device shown in FIG. The buffer solution is sent from the buffer solution tank (1) by the pump (2), and the sample (1 μl) is sent by the sampler (3).
Inject. The injected sample is divided into two by a three-way joint (5). One passes through the L-lactate oxidase column (6), hydrogen peroxide is produced from L-lactic acid, and the change in current value is detected by the hydrogen peroxide electrode (7). This electrode is the first electrode. One is D-LDH, L-LD
After passing through the H column (8), a conversion reaction of D-lactic acid and L-lactic acid occurs. After passing through the L-lactate oxidase column (9), a change in current value is detected at the hydrogen peroxide electrode (10). This is the second electrode. These columns and electrodes at 30 ℃
It is installed in the constant temperature bath (4). The change in current value at each electrode is detected by the detector (11). Further, the signal can be sent to the personal computer (14). The composition of the buffer solution is 100 mM sodium phosphate, 5
0 mM potassium chloride, 1 mM sodium azide, 5 mM
The pH is 7.0 including NAD. Pump flow rate is 1.3
ml / min, the flow rate at the first electrode is 0.7 ml / min,
The flow rate at the second electrode was 0.6 ml / min.

(5) Measurement of standard solution Distilled water 1, 2, 5, 10 mM L was added to the measuring device of (4).
-Lactic acid, 2, 5, 10, 20, 50 mM D-lactic acid was injected, and the current values obtained at the first and second electrodes were as shown in Table 1, and the following calibration curves were obtained. However, Y is the current value (n
A) and X are lactic acid concentrations (mM) in the sample. Since the first electrode (7) reacts only with L-lactic acid, the calibration curve 1 shows L-
This is a calibration curve for lactic acid only. At the second electrode (10), L-
A conversion reaction of lactic acid and D-lactic acid is performed, L-lactic acid decreases at a constant ratio when measuring L-lactic acid standard solution, and D-lactic acid remains at a constant ratio when measuring D-lactic acid standard solution. Is converted to L-lactic acid and detected. Since both D-lactic acid and L-lactic acid are detected, a calibration curve 2 which is a calibration curve of L-lactic acid and a calibration curve 3 which is a calibration curve of D-lactic acid are obtained. Calibration curve 1 1st electrode L-lactic acid calibration curve Y = 15.5X
+0.9 calibration curve 2 second electrode L-lactic acid calibration curve Y = 11.8X
-0.6 calibration curve 3 2nd electrode D-lactic acid calibration curve Y = 1.5X
-0.6

[0049]

[Table 1]

(6) Measurement of mixed sample A mixed solution of D-lactic acid and L-lactic acid was injected into the measuring device of (4). Table 2 shows the current values obtained at the first and second electrodes and the concentrations of D-lactic acid and L-lactic acid calculated from the calibration curve calculated by measuring the standard solution. To calculate the concentration of lactic acid in an unknown sample, firstly use the calibration curve 1 from the current value obtained at the first electrode.
The L-lactic acid concentration in the sample can be determined by From this concentration, the current value of L-lactic acid involved in the second electrode can be obtained from the calibration curve 2. From the current value obtained at the second electrode, L-
Since the remainder after subtracting the current value due to lactic acid is the current value due to D-lactic acid, the concentration of D-lactic acid can be obtained from this value and the calibration curve 3.

[0051]

[Table 2]

Example 2 (1) Production of L-lactate oxidase-immobilized column An L-lactate oxidase-immobilized column was prepared in the same manner as in Example 1. (2) Production of D-LDH and L-LDH-immobilized columns D-LDH and L-LDH-immobilized columns were prepared in the same manner as in Example 1. (3) Production of hydrogen peroxide electrode A hydrogen peroxide electrode was prepared in the same manner as in Example 1.

(4) Measuring device L-lactic acid and D-lactic acid are measured by the flow type measuring device shown in FIG. The buffer solution is sent from the buffer solution tank (21) by the pump 1 (22) and sent to the switching valve (23). This switching valve is a hexagonal valve, and A and B, C and D, and E and F are connected on the load side. On the inject side, B and C, D and E, and F and A are connected. Pump 1 (22) for A, L-lactate oxidase column for B (2
8) is arranged, C and F are D-LDH, L-LD
It is connected by the H column (25). Further, E is a sample inlet (24), and D is a pump 2 (26) for sucking the sample.
Connect.

On the load side, the buffer solution sent from the pump 1 (22) is sent to A, goes out of B and goes to the L-lactate oxidase column (28) in a thermostat (27) at 30 ° C. Liquid is sent. Sample is injected from E, D-LDH, L from F
-Transmitted to the LDH column (25), exiting C and connecting to D. When the pump 2 (26) is stopped in this state, a fixed amount of sample (about 100 μl) is added to D-LDH and LL.
The conversion reaction is carried out in the DH column (25). However, D-
In order to keep the reaction conditions for LDH and L-LDH constant, the sample was 5 mM NAD, 100 mM sodium phosphate, 50 mM.
pH containing mM potassium chloride, 1 mM sodium azide
Adjust to 7.0. Of course, if there is no NAD, the sample is D
-No reaction in LDH or L-LDH column. Therefore, by injecting a sample containing NAD and a sample not containing NAD, D-
It is possible to measure those reacted with the LDH and L-LDH columns and those not reacted. In addition, pump 2 (2
The stop time of 6) was 3 minutes.

When the valve is switched to the inject side, the buffer solution is sent from A to F, and a fixed amount of D-LDH,
The sample reacted in the L-LDH column is sent from C to B. When passing through the L-lactate oxidase column (28),
Hydrogen peroxide is generated from L-lactic acid, and the hydrogen peroxide electrode (2
The change in current value is detected by 9). The change in the current value at the electrodes is detected by the detector (30). Further, the signal can be sent to the personal computer (32).
The composition of the buffer solution pumped is 100 mM sodium phosphate, 50 mM potassium chloride, 1 mM sodium azide and has a pH of 7.0. Pump 1 flow rate is 1.0
It was ml / min.

(5) Measurement of standard solution Distilled water, 0.05 and 0.10 mM of L-lactic acid and 0.05 and 0.10 mM of D-lactic acid were injected into the measuring device of (4) and stopped for 3 minutes. Was measured. Each sample is NAD 5m
The measurement was carried out for those containing M and not containing M. The detected current value is shown in Table 3, and the following calibration curve was obtained. However, Y is the current value (nA), X is the lactic acid concentration in the sample (m
M). Calibration curve 1 without NAD L-lactic acid calibration curve Y = 679X
+0.08 Calibration curve 2 With NAD L-lactic acid calibration curve Y = 337X
-0.08 Calibration curve 3 With NAD D-lactic acid calibration curve Y = 330X
-0.03

[0057]

[Table 3]

(6) Measurement of mixed sample The mixed solution of D-lactic acid and L-lactic acid in the measuring device of (4) was measured for those containing NAD and not containing NAD. Table 4 shows the obtained current values and the concentrations of D-lactic acid and L-lactic acid calculated from the calibration curve calculated by the standard solution measurement.
The method for calculating the concentration of lactic acid in an unknown sample was the same as in Example 1.

[0059]

[Table 4]

[0060]

By using the measuring device of the present invention,
Now, D-lactic acid and L-lactic acid can be measured easily in a short time. In addition, L-LDH and D-LDH of the solution
Compared with the measurement of D-lactic acid and L-lactic acid using, the present invention allows the enzyme to be repeatedly used and facilitates the adjustment of the reaction conditions of the enzyme.

[Brief description of drawings]

FIG. 1 is a flow-type D-lactic acid L-used in Example 1.
It is a figure of a lactic acid measuring device.

FIG. 2 is a flow-type D-lactic acid L-used in Example 2.
It is a figure of a lactic acid measuring device.

FIG. 3 illustrates another aspect of the invention.

[Explanation of symbols]

 1 buffer tank 2 pump 3 sampler 4 thermostat tank 5 three-way joint 6 L-lactate oxidase-immobilized column 7 hydrogen peroxide electrode (first electrode) 8 D-LDH, L-LDH-immobilized column 9 L-lactate oxidase Immobilized column 10 Hydrogen peroxide electrode (second electrode) 11 Detector 12 Single board computer 13 RS232C code 14 Personal computer 15 Sampler control signal 16 Liquid feed pump control signal 17 Waste liquid 21 Buffer tank 22 Pump 1 23 Switching valve 24 Sample Injection port 25 D-LDH, L-LDH immobilized column 26 Pump 2 27 Constant temperature bath 28 L-Lactate oxidase immobilized column 29 Hydrogen peroxide electrode 30 Detector 31 Single board computer 32 Personal computer 33 RS232C code 34 Liquid transfer pump Control signal 35 abolished 41 buffer tank 42 pump 43 sampler 44 L-lactate oxidase-immobilized column 45 hydrogen peroxide electrode (first electrode) 46 D-LDH, L-LDH-immobilized column 47 L-lactate oxidase-immobilized column 48 peroxidation Hydrogen electrode (second electrode) 49 Constant temperature bath 50 Detector 51 Single board computer 52 Personal computer 53 RS232C code 54 Sampler control signal 55 Liquid feed pump control signal 56 Waste liquid

Continuation of front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location G01N 27/416

Claims (6)

[Claims] 1. An enzyme-immobilized body containing D-lactate dehydrogenase and L-lactate dehydrogenase, a mechanism for contacting nicotinamide adenine dinucleotide and a sample with the enzyme-immobilized body, and an L-lactic acid measurement mechanism. D-lactic acid and L- equipped
Lactic acid measuring device. 2. A mechanism for injecting a sample into a carrier to be fed from the upstream, a reactor containing an immobilized enzyme containing D-lactate dehydrogenase and L-lactate dehydrogenase, and L-lactic acid oxidation. Flow-type measurement of D-lactic acid and L-lactic acid, which comprises an enzyme-immobilized body and an electrode that detects an electrode active substance that increases or decreases when L-lactic acid in a sample is oxidized by L-lactic acid oxidase apparatus. 3. A mechanism having a mechanism for stopping the liquid feeding of the carrier or lowering the flow rate in the state where the enzyme-immobilized body containing D-lactate dehydrogenase and L-lactate dehydrogenase are in contact with the carrier containing the sample. Item 2. The measuring device according to item 2. 4. An electrode active substance that increases or decreases when L-lactic acid in a sample is oxidized by the L-lactate oxidase-immobilized body and L-lactate oxidase is detected on the upstream side of the reactor. The measuring device according to claim 2, wherein an electrode is provided. 5. A. The sample and nicotinamide adenine dinucleotide are brought into contact with an enzyme-immobilized body containing D-lactate dehydrogenase and L-lactate dehydrogenase, and D-lactic acid and L-in the sample
Measuring L-lactic acid after performing a conversion reaction with lactic acid, b. A step of measuring L-lactic acid in the sample,
Method for measuring lactic acid and L-lactic acid. 6. A. The sample and nicotinamide adenine dinucleotide are brought into contact with an enzyme-immobilized body containing D-lactate dehydrogenase and L-lactate dehydrogenase, and D-lactic acid and L-in the sample
Performing a conversion reaction with lactic acid, and b. A method for measuring D-lactic acid and L-lactic acid, which comprises a step of electrochemically detecting an electrode active substance that increases or decreases when L-lactic acid in a sample is oxidized by L-lactic acid oxidase. In step b, using L-lactic acid standard solution as a sample,
-The calibration curve 1 showing the relationship between the lactic acid concentration and the output value is obtained.-Using the L-lactic acid standard solution as a sample, the step a is performed, and then the step b is performed on the sample to show the relationship between the L-lactic acid concentration and the output value. The calibration curve 2 shown below is obtained, and step a is performed using a D-lactic acid standard solution as a sample, and then step b is performed for the sample to obtain a calibration curve 3 showing the relationship between the D-lactic acid concentration and the output value. In step b, a measurement sample is used as a sample, and an output value of 1 is measured, and when a measurement sample is used as a sample, step a is performed, and then step b is performed, an output value 2 is measured, and an output value 1, an output value 2 and a calibration curve are obtained. 1. Calculate the amount of D-lactic acid and the amount of L-lactic acid in the measurement sample from the calibration curve 2 and the calibration curve D-
Method for measuring lactic acid and L-lactic acid.