JP2016086680A - Enzyme immobilization body, analyzer equipped therewith, and measuring method of l-threonine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an enzyme immobilization body in which the substrate specificity of L-threonine dehydrogenase is improved, and which can be used stably and repeatedly, and also can quantify L-threonine simply and efficiently; and to provide a measuring device equipped with the immobilization body.SOLUTION: The invention provides an enzyme immobilization body in which L-threonine dehydrogenase and NADH oxidase are immobilized on the same membrane or carrier, and an analyzer of L-threonine equipped with a mechanism detecting an electrochemical active substance in the enzyme immobilization body and in the downstream of the above enzyme immobilization body.SELECTED DRAWING: Figure 1

Description

  The present invention relates to an enzyme-immobilized body that realizes substance conversion from L-threonine, and a measuring device including the immobilized body. Furthermore, it is related with the measuring method of L-threonine using this fixed body.

  L-threonine is classified as a completely essential amino acid that is not biosynthesized at all in the animal body. The required amount for a human adult male is 7 mg / kg body weight / day. Protein nutritional value is determined by the absolute amount and ratio of essential amino acids contained. Looking at the amino acid composition of cereals used in animal feeds, amino acids showing the smallest ratio of lysine, threonine, valine, isoleucine and the like among essential amino acids are called restricted amino acids. Threonine is a component that is likely to be a restricted amino acid. Therefore, it is possible to increase the production of animal protein by adding these amino acids to the feed (edited by Ajinomoto Co., Inc., “Amino Acid Handbook”, first edition, Industrial Research Co., Ltd., April 2003, 20-27, 171). page). Various production methods such as separation and purification methods from natural products, chemical synthesis methods, and microbial fermentation methods have already been developed.

  While these production methods have been developed, it is not easy to quantify L-threonine simply. Generally, a high performance liquid chromatographic method, particularly an amino acid analyzer is used. In this method, sample pre-treatment such as filtration, decolorization, and sterilization is essential, and analysis requires one hour or more, and the apparatus itself is expensive and maintenance is complicated. It cannot be easily used on a daily basis.

  As a colorimetric method that can be carried out with a relatively inexpensive apparatus, threonine is oxidized with periodate to form acetaldehyde, aerated and supplemented with an acidic sodium sulfite solution, and p-hydroxydiphenol is allowed to act on red. There is a method of Neidig-Hess that develops and quantifies purple color (Shinji Funayama, “Amino Acid”, 1st edition, Tokyo Denki University Press, July 2009, page 541). This colorimetric method also requires a multi-stage analysis operation, and is not easy.

For these methods, a sample containing L-threonine using L-threonine dehydrogenase derived from Cupriavidus necator, the enzyme and the coenzyme NAD ⁺ are mixed, and the amount of NADH produced or A method for quantifying α-amino-β-ketobutyric acid has been proposed (Patent Document 1).

  In Patent Document 1, a method of measuring the generated NADH by a change in absorbance at 340 nm, a method of measuring fluorescence of NADH, a method of measuring the color development in the visible region by reacting phenazine methosulfate as a mediator, or using diaphorase There is exemplified a method of performing quantitative determination of L-threonine by oxidizing NADH and quantifying by reducing the color of the dye and coloring.

  However, the property of L-threonine dehydrogenase is that the oxidation rate of L-threonine is slow, and it takes several minutes to several tens of minutes to proceed the reaction quantitatively.

  Further, when the secondary hydroxyl group of L-threonine is oxidized, α-amino-β-ketobutyric acid is obtained. Although this compound is highly reactive and can be a starting material for chemical raw materials, it is an unstable compound and spontaneously decomposes into aminoacetone and carbon dioxide. When this compound is obtained or used as a synthetic raw material, there is a high possibility that spontaneous decomposition will occur preferentially unless the oxidation reaction is carried out quickly and efficiently. It may be possible to efficiently produce unstable α-amino-β-ketobutyric acid by changing the utilization form of the enzyme. Improving this efficiency opens the way to oxidize L-threonine quantitatively, and as a result, improvements in quantitative sensitivity and accuracy are expected.

  In Patent Document 1, L-threonine dehydrogenase has been isolated from microorganisms other than Capriavidas Necatol, but it has been pointed out that these enzymes have poor substrate specificity and respond to L-serine. In conclusion, even if L-threonine dehydrogenase is obtained from various microorganisms, it is difficult to use it for analytical purposes.

  When using an enzyme for analytical purposes, the method of using it as an immobilized enzyme electrode is considered to be the most cost effective and can be easily used. Patent Document 1 also mentions an enzyme sensor, but examples include those that use diaphorase as an immobilization body and those that directly oxidize NADH on the electrode surface.

Japanese Patent No. 4997822

  The present invention is provided with an enzyme-immobilized body in which the substrate specificity of L-threonine dehydrogenase is improved, can be stably and repeatedly used, and L-threonine can be quantified more simply and efficiently. It aims at providing a measuring device. It is another object of the present invention to provide a measurement method that is excellent in substrate specificity and that can quantitate L-threonine simply and efficiently.

  The present invention discloses an enzyme-immobilized body in which L-threonine dehydrogenase and NADH oxidase are immobilized on the same membrane or carrier (hereinafter also referred to as “L-threonine dehydrogenase / NADH oxidase-immobilized body”). To do.

  In particular, L-threonine dehydrogenase is a microorganism belonging to the genus Pyrococcus, Thermus, Clostridium, Flavobactrerium, Meiothermus, and Thermoplasma. It is preferably derived from at least one microorganism selected from the group consisting of

  In addition, an L-threonine analyzer comprising the above enzyme-immobilized body and a mechanism for detecting an electrochemically active substance downstream of the immobilized body is disclosed.

  That is, a mechanism for forming a buffer flow, a mechanism for injecting a sample into the buffer flow, the enzyme-immobilized body downstream of the injection mechanism, and an electrochemically active substance downstream of the immobilized body And an L-threonine analyzer equipped with a mechanism for detecting.

  And a step of detecting L-threonine in a sample using an enzyme-immobilized body in which L-threonine dehydrogenase and NADH oxidase are immobilized on the same membrane or carrier, and a mechanism for detecting an electrochemically active substance. A method for measuring L-threonine in a specimen is disclosed.

In particular, it is preferable that a buffer solution containing NAD ⁺ is sent to the enzyme-immobilized body.

  By immobilizing L-threonine dehydrogenase and NADH oxidase on the same membrane or carrier, the oxidation rate of L-threonine can be remarkably improved and excellent quantitative properties are obtained. Furthermore, the substrate specificity could be improved by carrying out the immobilization using various L-threonine dehydrogenases.

It is the schematic of a flow type measuring apparatus. It is a graph which shows the relationship between pH and a conversion rate (L-threonine dehydrogenase derived from Meiothermus sylvanus). It is a graph which shows the relationship between pH and conversion rate (Thermoplasma acidophilum origin L-threonine dehydrogenase). It is a graph which shows the relationship between the correlation coefficient (L-threonine dehydrogenase derived from Meiothermus sylvanus) of pH and a calibration curve. It is a graph which shows the relationship between the correlation coefficient (Thermoplasma acidophilum origin L-threonine dehydrogenase) of pH and a calibration curve. It is a graph which shows the relationship between NAD ⁺ density | concentration and a conversion rate. It is a graph which shows the relationship between the correlation coefficient of NAD ⁺ density | concentration and a calibration curve. It is a graph which shows a L-threonine calibration curve.

  Hereinafter, the present invention will be described in detail.

  L-threonine dehydrogenase (EC 1.1.1.103) is isolated from bacteria and the like. This enzyme catalyzes the following reaction (1).

This reaction is biased to the left, and the reduction reaction in the direction of producing L-threonine from the oxidized substrate and NADH, which is a coenzyme, is carried out rapidly, but the enzyme is simply mixed with L-threonine and NAD ^+. The oxidation reaction proceeds slowly even if it is contacted with. Thus, the oxidation reaction could proceed by simultaneously immobilizing NADH oxidase and quickly returning the produced NADH to NAD ⁺ .

  NADH oxidase (EC 1.6.3.1) catalyzes the following reaction (2).

  NADH produced by the L-threonine oxidation reaction by L-threonine dehydrogenase is immediately oxidized by NADH oxidase. Since NADH oxidase suppresses a reaction that oxidizes a high concentration of NADH in the presence of NAD, it is essential to coexist with coenzyme FAD. However, in recent years, NADH oxidase that does not require activation by FAD and specifically oxidizes NADH has been isolated from microorganisms of the genus Brevibacterium.

  The L-threonine dehydrogenase and NADH oxidase in the present invention may be derived from any organism. In this specification, the enzyme derived from a certain organism (microorganism, animal, plant) may be an enzyme itself produced by the organism, and further, one or more amino acids are substituted in the amino acid sequence of the enzyme. Variants obtained by addition, deletion and insertion are widely included.

  As methods for immobilizing L-threonine dehydrogenase and NADH oxidase, methods known as protein immobilization methods such as physical adsorption method, ion binding method, inclusion method, and covalent bond method can be used. Excellent in properties and desirable. As a method for covalently binding a protein, various methods such as a method using a compound having an aldehyde group such as formaldehyde, glyoxal, and glutaraldehyde, a method using a polyfunctional acylating agent, a method of crosslinking a sulfhydryl group, and the like are used. it can.

  In the present invention, the immobilization of L-threonine dehydrogenase and NADH oxidase is preferably carried out in a mixed state of L-threonine dehydrogenase and NADH oxidase at the same time on the same membrane or carrier.

  The enzyme-immobilized body can be immobilized on a membrane, or can be immobilized on an insoluble carrier and packed in a column reactor. In addition, other types of enzymes, proteins such as gelatin and serum albumin, and synthetic polymers such as polyallylamine and polylysine coexist at the time of immobilization to change the properties of the immobilized enzyme, that is, membrane strength, substrate permeability, etc. You can also

  As the carrier for immobilizing the enzyme on an insoluble carrier, diatomaceous earth, activated carbon, alumina, titanium oxide, silica gel as an inorganic carrier, crosslinked starch particles as an organic carrier, cellulose-based polymer, chitin, chitosan derivative, etc. A known carrier can be used. Among these, an inorganic carrier is particularly preferable for ensuring a stable calibration curve with excellent pressure resistance.

  L-threonine dehydrogenase can be obtained from a wide range of microorganisms such as hyperthermophilic bacteria, moderately thermophilic bacteria, and thermophilic bacteria. In general, when an enzyme purified from a hyperthermophilic bacterium is immobilized, the heat resistance of the immobilized body is good. However, the most suitable L-threonine dehydrogenase to be immobilized at the same time varies depending on the optimum temperature of the enzyme used at the same time. For example, the optimum temperature of NADH oxidase obtained from a Bacillus genus that is a thermophilic bacterium is around 40 ° C., and the optimum temperature is slightly higher in the immobilized body, but it is desirable to use in the range of 30 to 45 ° C. . In such a case, when L-threonine dehydrogenase derived from a hyperthermophilic bacterium is used, the reaction rate of the step of producing NADH is slowed, and the overall efficiency may be lowered. On the other hand, use of moderately thermophilic or thermophilic enzymes is more preferable because the maximum speed is often obtained at the operating temperature of NADH oxidase, and an economic effect such as reduction of the amount of immobilization can be expected.

  The amount of enzyme immobilized varies depending on the particle size of the carrier used for analysis, the contact time of the sample, and the like. In the case of a reactor type using an immobilized column, L-threonine dehydrogenase is 1 to 1 in one column. It is desirable to fix 1000 units, more preferably 5 to 500 units. Similarly, NADH oxidase should be immobilized at 1-1000 units, more preferably at 2-100 units. For any enzyme, if the activity is too low, the progress of the reaction is slow and the predetermined analytical sensitivity is often not obtained.

In this specification, the amount of L-threonine dehydrogenase enzyme is defined as 1 U of the amount of enzyme that converts 1 μmol of L-threonine to 1 μmol of α-amino-βbutyric acid per minute at 37 ° C. The amount of NADH oxidase enzyme is 1 U at 37 ° C. to convert 1 μmol NADH to 1 μmol NAD ⁺ per minute.

  The optimum pH of the L-threonine dehydrogenase immobilized product is 6.0 to 10.0, more preferably 7.5 to 8.5. This coincides with the optimum pH of the NADH oxidase used as the immobilized body at the same time, and the pH when using the simultaneously immobilized body of the present invention is preferably 7.5 to 8.5. In this pH range, a phosphate buffer solution, a citrate-phosphate buffer solution, a pyrophosphate buffer solution, a borate buffer solution and the like are preferable because they have a strong buffer capacity.

  Moreover, as the characteristic of the L-threonine dehydrogenase / NADH oxidase immobilized product of the present invention, the optimum temperature is around 40 ° C. The optimum temperature means the temperature at which the maximum rate is obtained by increasing the temperature as long as the reaction rate continues to increase. In actual analysis, it is often used to prevent the measurement results from being affected by fluctuations in room temperature and to increase the sensitivity of the electrode for detecting electrochemically active substances such as hydrogen peroxide generated by enzymatic reactions. The temperature to be applied is 30-50 ° C.

  Hydrogen peroxide can be measured directly or indirectly by a known method. An electrochemical technique such as amperometry is preferably used for highly sensitive measurement of hydrogen peroxide.

  In order to advance the reaction by contacting the sample with the immobilized enzyme for a certain period of time, a batch method in which the reaction is caused while stirring the sample liquid for a certain period of time is possible, but in order to carry out a more accurate measurement. It is desirable to use flow based measurements. Of course, since the surface area of the carrier to be immobilized is constant, the amount of enzyme that can be immobilized is limited, and the cost increases if the amount of enzyme to be immobilized is increased. Therefore, it is desirable to efficiently convert L-threonine to hydrogen peroxide with as low an enzyme amount as possible. As a method therefor, increasing the contact time between the enzyme-immobilized body and the sample can be mentioned. In order to increase the contact time, it is preferable to reduce the particle size of the carrier to increase the contact area, to reduce the flow rate, or to stop the liquid feeding for a certain time while the enzyme-immobilized body is in contact with the sample.

  In the present invention, a flow type apparatus capable of performing measurement with higher accuracy is disclosed. One preferred embodiment of the present invention shown in FIG. 1 comprises a buffer bottle (1), a pump (2), and an autosampler (3) for injecting a sample. The L-threonine dehydrogenase / NADH oxidase-immobilized product (5) is placed downstream of the autosampler (3). An electrode capable of detecting the electrochemically active substance concentration is disposed downstream thereof. In this case, it is a hydrogen peroxide electrode (6). A change in the current value of the hydrogen peroxide electrode (6) is converted into a voltage change by the current-voltage converter (7), digitized by the board computer (8), and sent to the personal computer (10) for analysis. The waste liquid used for the analysis is discharged into a waste liquid bottle (9).

The buffer solution to be passed through this apparatus is not particularly limited, but is selected so that the pH of the enzyme-immobilized body becomes high (for example, pH 7.5 to 8.5). The buffer solution contains NAD ^{+ and} may contain FAD, antibacterial agent, surfactant and the like, if necessary.

The concentration of NAD ⁺ in the buffer is preferably 10 μM or more, more preferably 100 to 1000 μM. Although it can be used even at a concentration higher than 1000 μM, addition of an unnecessarily high concentration causes an increase in analysis cost. When used at a concentration lower than 10 μM, it is necessary to lower the measurement range of L-threonine. Further, not only free NAD ⁺ but also more stable NAD ⁺ derivatives can be used. The concentration of FAD in the buffer is preferably 10 to 1000 μM, more preferably 500 to 1000 μM. No effect is observed at very low concentrations, but coenzymes are more expensive than inorganic salts used in buffers, and adding unnecessarily high concentrations causes an increase in analysis costs.

  The temperature of the constant temperature bath (4) is 25 to 40 ° C, more preferably 30 to 39 ° C. The flow rate is 0.1 to 2.0 mL / min, more preferably 0.5 to 1.5 mL / min.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

[Example]
(1) Method for producing L-threonine dehydrogenase / NADH oxidase co-immobilized column derived from Meiothermus silvanus After immersing 75 mg of aminosilanized silica gel carrier in 5% glutaraldehyde for 1 hour, thoroughly wash with distilled water. Finally, it was replaced with 100 mM sodium phosphate buffer at pH 7.0, removing this buffer as much as possible. 250 μl of a solution prepared by dissolving 37 units / ml of L-threonine dehydrogenase derived from Meiothermus sylvanus in a pH 7.0, 100 mM sodium phosphate buffer solution on this formylated silica gel carrier, Bacillus in 100 mM sodium phosphate buffer solution -1 ml of a solution dissolved in a concentration of 1 unit / ml of NADH oxidase derived from Bacillus licheniformis was simultaneously contacted and allowed to stand at 0-4 ° C for 1 day for immobilization. This enzyme-immobilized carrier was packed in a column having an inner diameter of 3.5 mm and a length of 15 mm.

(2) Manufacturing method of L-threonine dehydrogenase / NADH oxidase co-immobilized column derived from Thermoplasma acidophilum 75 mg of aminosilanized silica gel carrier was immersed in 5% glutaraldehyde for 1 hour, and then thoroughly washed with distilled water Finally, the pH 7.0 was replaced with 100 mM sodium phosphate buffer, and this buffer was removed as much as possible. 250 μl of pH 7.0, 100 mM sodium phosphate buffer solution dissolved in thermoplasma acidophilum L-threonine dehydrogenase at a concentration of 6.7 units / ml in this formylated silica gel carrier, pH 7.0, 100 mM sodium phosphate buffer The solution is simultaneously brought into contact with 1 ml of a solution of NADH oxidase derived from Bacillus licenformis 1 unit / ml and allowed to stand at 0 to 4 ° C. for 1 day for immobilization. This enzyme-immobilized carrier was packed in a column having an inner diameter of 3.5 mm and a length of 15 mm.

(3) Method for Producing Hydrogen Peroxide Electrode A hydrogen peroxide electrode was obtained by depositing a noble metal on a glass plate by vapor deposition. Three noble metals platinum, platinum, and silver were vapor-deposited on a non-alkali glass substrate having a thickness of 0.7 mm. Silver was used as a reference electrode, one platinum was used as a working electrode, and the other was used as a counter electrode for supplying electrons. Cellulose acetate was spin-coated at a thickness of 1 μm on the noble metal thin film. Cellulose acetate permeates low molecular weight compounds such as hydrogen peroxide, and molecular weight such as ascorbic acid is relatively large, and compounds that are oxidized at the same potential as hydrogen peroxide reach the platinum working electrode surface. Disturb.

  A noble metal thin film formed on the glass plate thus prepared was incorporated in a flow cell, and a voltage of +0.6 V was applied to the platinum electrode with respect to the silver chloride silver electrode.

(4) Measuring apparatus FIG. 1 is a flow type measuring apparatus equipped with the aforementioned L-threonine dehydrogenase / NADH oxidase simultaneous immobilization product. The buffer solution was fed from the buffer solution tank (1) by the pump (2), and 4 μl of the sample was injected from the autosampler (3). The fed sample passes through the L-threonine dehydrogenase / NADH oxidase simultaneous immobilization column (5) installed in the thermostat (4), and hydrogen peroxide is generated from L-threonine. The generated hydrogen peroxide passes through the downstream hydrogen peroxide electrode (6) and causes a change in current value.

  The change in the current value is converted into a voltage change by the current-voltage converter (7), digitized by the board computer (8), and sent to the personal computer (10) for analysis. The waste liquid used for the analysis is discharged into a waste liquid bottle (9). The flow rate of the buffer solution was 1.0 ml / min, and the temperature of the thermostatic bath was 37 ° C.

(5) Conversion rate calculation method L-threonine standards prepared at 0, 1, 2, and 5 mM, respectively, were injected into the measuring apparatus, and the detected current values of the standards at each concentration were recorded. After plotting the detected current value on the graph so that the horizontal (X) axis is the standard concentration and the vertical (Y) axis is the detected current value, the relationship between the concentration and the detected current value is represented by the least square method. A calibration curve was prepared. At this time, the calibration curve is a linear line of Y = aX + b, and the slope a represents the current value per unit concentration (1 mM). For hydrogen peroxide and NADH, a calibration curve representing the relationship between the concentration and the detected current value was prepared in the same manner, and the slope was calculated.

  The L-threonine / NADH conversion rate is a ratio between the slope of the L-threonine calibration curve and the slope of the NADH calibration curve, and is a value representing the conversion efficiency from L-threonine to NADH. This value is an index indicating the activity of L-threonine dehydrogenase in the column.

  The L-threonine / hydrogen peroxide conversion rate is a ratio of the slope of the L-threonine calibration curve and the slope of the hydrogen peroxide calibration curve, and is a value representing the conversion efficiency from L-threonine to hydrogen peroxide. This value is an index indicating the overall activity of L-threonine dehydrogenase and NADH oxidase in the column.

(6) Buffer used The pH of the buffer used was examined between 6.5 and 9.0. The results are shown in FIGS. When the pH was 7.5 to 8.5, the L-threonine / hydrogen peroxide conversion rate, the L-threonine / NADH conversion rate, and the correlation coefficient of the calibration curve were improved.

(7) NAD ⁺ concentration in buffer solution The NAD ⁺ concentration added to the buffer solution was examined between 0.01 mM and 1.00 mM. The results are shown in FIGS. When the NAD ⁺ concentration was 0.10 to 1.00 mM, the L-threonine / hydrogen peroxide conversion rate, the L-threonine / NADH conversion rate, and the correlation coefficient of the calibration curve were improved.

(8) Linear region of calibration curve A calibration curve for L-threonine was prepared using a 50 mM pyrophosphate buffer (pH 8.0) containing 50 mM potassium chloride, 0.5 mM NAD ⁺ , and 50 μM FAD. The results are shown in FIG. The correlation coefficient of 0.9999 was maintained until the L-threonine concentration was 10 mM.

(9) Examination of substrate specificity The substrate specificity of this immobilized body was examined. Various amino acids prepared to 1 mM were injected into the measuring apparatus, and the detected current value for each amino acid was recorded. The reaction rate to other amino acids was calculated with the current value of L-threonine as 100%. The results are shown in Table 1. Other than L-threonine, there was almost no response.

[Comparative example]
(1) Separation type immobilization column production method In the method described in Examples (1) and (2), only L-threonine dehydrogenase is immobilized. Separation type L-threonine dehydrogenase immobilization column, NADH oxidase only Is used as a separation-type NADH oxidase-immobilized column. What connected these in series with the Teflon (trademark) tube was used for the comparison experiment.

(2) Comparison with L-threonine dehydrogenase / NADH oxidase co-immobilized body Using the apparatus of Example (4), 4 μl of 0, 1, 2, 5 mM L-threonine was injected to obtain detection values. . From the detected values, a calibration curve when using the simultaneously immobilized body and a calibration curve when using a separation column were prepared. The L-threonine / hydrogen peroxide conversion rate and the L-threonine / NADH conversion rate were calculated from the slope of the calibration curve. The results are shown in Tables 2-3. By simultaneously immobilizing L-threonine dehydrogenase and NADH oxidase, the L-threonine / hydrogen peroxide conversion rate and the L-threonine / NADH conversion rate were improved.

  The present invention is useful for a substance conversion reaction using L-threonine dehydrogenase, and can be applied to quantitative analysis of L-threonine in samples such as blood, fermentation broth, and food.

DESCRIPTION OF SYMBOLS 1 Buffer bottle 2 Liquid feed pump 3 Autosampler 4 Constant temperature bath 5 Immobilization column reactor 6 Hydrogen peroxide electrode 7 Current-voltage converter 8 Board computer 9 Waste liquid bottle 10 Personal computer

Claims (6)

  An enzyme-immobilized product in which L-threonine dehydrogenase and NADH oxidase are immobilized on the same membrane or carrier.   The L-threonine dehydrogenase is derived from at least one microorganism selected from the group consisting of microorganisms belonging to the genus Pyrococcus, Thermus, Clostridium, Flavobacterium, Meiothermus, and Thermoplasma. The enzyme-immobilized body according to claim 1.   3. An L-threonine analyzer comprising the enzyme-immobilized body according to claim 1 and a mechanism for detecting an electrochemically active substance downstream of the immobilized body.   A mechanism for forming a buffer flow, a mechanism for injecting a sample into the buffer flow, the enzyme-immobilized body according to claim 1 or 2 downstream of the injection mechanism, and an electric downstream of the immobilized body. An apparatus for analyzing L-threonine having a mechanism for detecting a chemically active substance.   An enzyme-immobilized body in which L-threonine dehydrogenase and NADH oxidase are immobilized on the same membrane or carrier, and a step of detecting L-threonine in a specimen using a mechanism for detecting an electrochemically active substance. A method for measuring L-threonine in a specimen. The measurement method according to claim 5, wherein a buffer solution containing NAD ⁺ is sent to the enzyme-immobilized body.