Preparation method of nicotinamide adenine dinucleotide

Abstract

The invention belongs to the technical field of electrochemistry and chemical engineering, and particularly relates to a preparation method of nicotinamide adenine dinucleotide. The nicotinamide adenine dinucleotide can be efficiently prepared at a high yield under a low negative voltage by constructing a reaction system and utilizing cooperation between an electrode material and an electrochemical potential. The composite metal electrode is prepared by calcining metal and carbon, so that the yield of nicotinamide adenine dinucleotide is increased. By selecting the foam metal, diffusion and adsorption of NAD * and H * in the electrode material are facilitated, and more active sites are provided for charge transfer. According to the application, the carbon nanoparticles are introduced into the reaction liquid, and more active sites are provided to improve the electron transfer efficiency, so that the yield of nicotinamide adenine dinucleotide is further improved. Tests show that titanium foam metal is used as a working electrode, carbon nanoparticles are added into a reaction solution, and the yield is up to 99.3% after the reaction is carried out for 5s under the low negative voltage of-0.7 V.

Description

Technical Field

[0001] The present application belongs to the field of electrochemical chemical technology, and specifically relates to a method for preparing nicotinamide adenine dinucleotide. Background Art

[0002] Enzymatic reactions catalyzed by oxidoreductases usually require certain types of cofactors. One of the most commonly used cofactors is nicotinamide adenine dinucleotide (1,4-NADH), which plays an important role in the shuttling of electrons and hydrogen catalyzed by the enzyme. In recent years, due to the further promotion of the industrial application of oxidoreductases, the demand for coenzymes and coenzyme regeneration systems has increased. Therefore, the invention of a low-cost, high-efficiency and green coenzyme regeneration system has important theoretical significance and practical value.

[0003] In the redox reaction, 1,4-NADH acts as a hydrogen and electron donor, and NAD + As acceptors of hydrogen and electrons, they participate in physiological processes such as respiration, photosynthesis, and alcohol metabolism. As coenzymes for many redox reactions in organisms, they are used to participate in life activities and transform into each other. This is the basic principle of coenzyme regeneration. Common 1,4-NADH regeneration methods include enzymatic, chemical, and photochemical methods. Based on the characteristics of simple and convenient operation, green and pollution-free electrochemical methods, the reaction process can be monitored by an electrochemical workstation to provide oxidized / reduced coenzymes for various redox enzyme catalytic reactions.

[0004] Electrochemical regeneration of NADH is mainly carried out in two steps. First, NAD + Under negative overpotential (around -1.1V), unstable NAD is generated. * Free radicals, the second step is NAD * The free radical protonates, and then obtains an electron and hydrogen ion to reduce to generate 1,4-NADH. However, the existing electrochemical methods for regenerating 1,4-NADH require inconvenient electrode preparation and large negative reduction potential. For example, NADH is prepared electrochemically using glassy carbon electrodes, but on the one hand, the negative voltage of -2.4V is relatively high, and long-term electrocatalytic reactions will inactivate the enzyme in the enzymatic reaction; on the other hand, NAD * The efficiency of protonation of free radicals in preparing nicotinamide adenine dinucleotide is low, making it difficult to achieve stable cyclic regeneration of the coenzyme. Summary of the invention

[0005] In order to solve the above technical problems, the present application provides a method for preparing nicotinamide adenine dinucleotide, which is specifically achieved through the following technical solutions: A method for preparing nicotinamide adenine dinucleotide, using oxidized nicotinamide adenine dinucleotide (NAD+ ) as raw material, a metal electrode as a working electrode, glassy carbon as an auxiliary electrode, and a Pt|Hg(I)Hg2Cl2(s)|KCl electrode as a reference electrode to electrochemically synthesize nicotinamide adenine dinucleotide; the metal electrode contains one or more metals selected from platinum, palladium, copper and titanium; the conditions for the electrochemical synthesis are: voltage -0.5~-1.8 V.

[0006] Preferably, the metal electrode is palladium metal, and the conditions for the electrochemical synthesis are: voltage -1.4 V, time 3s.

[0007] Preferably, the metal is foamed metal.

[0008] Preferably, the metal electrode is titanium foam, and the conditions for the electrochemical synthesis are: voltage -0.7V, time 5 to 60s.

[0009] Preferably, the metal electrode is foamed copper, and the conditions for the electrochemical synthesis are: voltage -1.1V, time 6s.

[0010] Preferably, the reaction solution of the electrochemical synthesis contains carbon nanoparticles.

[0011] Preferably, the metal electrode is titanium foam; the conditions for the electrochemical synthesis are: voltage -0.7 V, time 3s.

[0012] Preferably, the metal electrode is prepared by calcining the metal and carbon; the conditions of the electrochemical synthesis are: voltage -0.7 V, time 5s.

[0013] Preferably, the electrochemical synthesis conditions also include: temperature 35° C., pressure 1.01 MPa, and rotation speed 600 rpm.

[0014] Compared with the prior art, this application has the following beneficial effects: This application constructs a reaction system and utilizes the coordination between electrode materials and electrochemical voltage to prepare nicotinamide adenine dinucleotide with high efficiency and high yield at low negative voltage. After testing, it was found that in the reaction system of this application, when palladium gold was used as the working electrode, the yield of nicotinamide adenine dinucleotide in a 3s reaction at a voltage of -1.4V could reach 93%.

[0015] This application further improves the yield of nicotinamide adenine dinucleotide by preparing a composite metal electrode by calcining metal and carbon. Specifically, taking the composite metal electrode prepared by calcining titanium metal and carbon as an example, the yield of nicotinamide adenine dinucleotide is as high as 99.4% under a low negative voltage of -0.7V for 5S. In addition, in the subsequent catalytic process, the superior conductivity of carbon itself can be used to promote the electron transfer process in the catalytic reaction and improve the catalytic efficiency.

[0016] The present invention is beneficial to NAD by selecting foam metal, three-dimensional porous structure and branched wall structure of foam metal. * and H * Diffusion and adsorption in the electrode material increase the effective area of ​​the electrode, providing more active sites for charge transfer. According to tests, when titanium foam is used as the working electrode, the yield of 3s nicotinamide adenine dinucleotide can reach 95.2% at a voltage of -0.7V.

[0017] This application introduces carbon nanoparticles into the reaction solution, improves the electron transfer efficiency by providing more active sites, and further improves the yield of nicotinamide adenine dinucleotide. After testing, titanium foam metal is used as the working electrode, and carbon nanoparticles are added to the reaction solution, and the yield is as high as 99.3% at a low negative voltage of -0.7V for 5S. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to clearly illustrate the embodiment, the drawings of the figures are briefly introduced below: Figure 1 Schematic diagram of the reaction equation for electrochemical regeneration of reduced nicotinamide adenine dinucleotide.

[0019] Figure 2 This is a graph showing the yield of 1,4-NADH under different potential conditions when using metal platinum and palladium electrodes as working electrodes during the 5S reaction of Examples 1 and 2. Figure 3 The graph is a graph of the yield of 1,4-NADH at different catalytic times in Examples 1 and 2 using metal platinum and palladium electrodes as working electrodes.

[0020] Figure 4 This is a graph showing the yield of 1,4-NADH under different potential conditions when the reaction is 5s in Examples 3 and 4, using foam copper and foam titanium electrodes as working electrodes. Figure 5 The yield of 1,4-NADH at different catalytic times is shown in Examples 3 and 4 using foam copper and foam titanium electrodes as working electrodes.

[0021] Figure 6This is a graph showing the yield of 1,4-NADH under different potential conditions when the reaction is 5s in Examples 5 and 6, using foamed titanium-carbon nanopowder and titanium metal carbon composite electrode as working electrodes. Figure 7 The graphs show the yield of 1,4-NADH at different times when using foamed titanium-carbon nanopowder and titanium metal carbon composite electrode as working electrodes in Examples 5 and 6. DETAILED DESCRIPTION

[0022] The present application will be further described below in the form of specific embodiments. Those of ordinary skill in the art will be able to implement the present application based on these descriptions. In addition, the embodiments of the present application involved in the following description are generally only embodiments of a part of the present application, rather than all embodiments. Therefore, based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative work should fall within the scope of protection of the present application.

[0023] The configuration method of the reaction system in the following examples is as follows: a potentiostat (Chenhua electrochemical workstation, CHI600C), a constant temperature water bath stirrer and a homemade three-electrode electrochemical cell were used to study the oxidation form (NAD + ) was electrochemically reduced to 1,4-NADH. Phosphate buffer (PBS, 0.1 mol / L, pH 7.5) was used to prepare 5 mmol / L NAD in a volume of 100 mL. + Solution. The geometric surface area of ​​the pre-processed 2 The metal electrode is used as the working electrode, and glassy carbon and Pt|Hg(I)Hg2Cl2(s)|KCl are used as the counter electrode and reference electrode. The entire reaction system is then placed in a constant temperature water bath stirrer at a temperature of 35°C (considering that the optimum temperature of enzymes in most biocatalysis is 35°C). The method of the pre-treatment is as follows: the metal sheet electrode, the foam metal electrode and the metal-carbon composite electrode are soaked in anhydrous ethanol for 24 hours to remove the insoluble impurities in the electrode sheet. Take out the electrode and rinse the ethanol with deionized water, use ultrasound to remove the residual impurities on the electrode surface, and then place the electrode in an oven to completely evaporate the water and ethanol.

[0024] Example 1 This embodiment discloses a method for preparing nicotinamide adenine dinucleotide using platinum as a working electrode, which specifically includes the following steps: using PBS buffer (0.1M, pH 7.5) to prepare 5 mmol / L NAD in a volume of 100 mL +solution, place the reactor on an electromagnetic stirring heater at 35°C and 600r / min, clamp metal platinum into the working electrode of the electrochemical workstation, clamp glassy carbon into the auxiliary electrode of the electrochemical workstation, and connect Pt|Hg(I)Hg2Cl2(s)|KCl to the reference electrode.

[0025] Set the electrochemical workstation to use different voltages to react for 5S, refer to the attached Figure 2 , select the best reaction potential, apply constant voltage to react for 60S, take samples intermittently, and use a Biowave UV-Vis spectrophotometer to determine the yield of regenerated active 1,4-NADH. Figure 3 By observing Figure 2 and 3 It can be found that when metal platinum is used as the working electrode, the optimal reaction parameters are voltage -1.4V and time 10s.

[0026] Example 2 This embodiment discloses a method for preparing nicotinamide adenine dinucleotide using palladium as a working electrode, which specifically includes the following steps: using PBS buffer (0.1M, pH 7.5) to prepare 5 mmol / L NAD in a volume of 100 mL + solution, place the reactor on an electromagnetic stirring heater at 35°C and 600r / min, clamp palladium and gold into the working electrode of the electrochemical workstation, clamp glassy carbon into the auxiliary electrode of the electrochemical workstation, and connect Pt|Hg(I)Hg2Cl2(s)|KCl to the reference electrode.

[0027] Set the electrochemical workstation to use different voltages to react for 5S, refer to the attached Figure 2 , select the best reaction potential, apply constant voltage to react for 60S, take samples intermittently, and use a Biowave UV-Vis spectrophotometer to determine the yield of regenerated active 1,4-NADH. Figure 3 By observing Figure 2 and 3 It can be found that when metal palladium is used as the working electrode, the yield of 3s nicotinamide adenine dinucleotide in the -1.4V voltage reaction can reach 93%.

[0028] Example 3 This embodiment discloses a method for preparing nicotinamide adenine dinucleotide using a three-dimensional porous foam metal electrode, which specifically includes the following steps: using PBS buffer (0.1M, pH 7.5) to prepare 5 mmol / L NAD in a volume of 100 mL. +solution, place the reactor on an electromagnetic stirring heater at 35°C and 600r / min, clamp the foam copper into the working electrode of the electrochemical workstation, clamp the glassy carbon into the auxiliary electrode of the electrochemical workstation, and connect Pt|Hg(I)Hg2Cl2(s)|KCl to the reference electrode.

[0029] Set the electrochemical workstation to use different voltages to react for 5S, refer to the attached Figure 4 , select the best reaction potential, apply constant voltage to react for 60S, take samples intermittently, and use a Biowave UV-Vis spectrophotometer to determine the yield of regenerated active 1,4-NADH. Figure 5 By observing Figure 4 and 5 It can be found that when copper foam is used as the working electrode, the yield of 6s nicotinamide adenine dinucleotide can reach 80.5% at a voltage of -1.1V.

[0030] Example 4 This embodiment discloses a method for preparing nicotinamide adenine dinucleotide using a three-dimensional porous foam metal electrode, which specifically includes the following steps: using PBS buffer (0.1M, pH 7.5) to prepare 5 mmol / L NAD in a volume of 100 mL. + solution, place the reactor on an electromagnetic stirring heater at 35°C and 600r / min, clamp the foam titanium into the working electrode of the electrochemical workstation, clamp the glassy carbon into the auxiliary electrode of the electrochemical workstation, and connect Pt|Hg(I)Hg2Cl2(s)|KCl to the reference electrode.

[0031] Set the electrochemical workstation to use different voltages to react for 5S, refer to the attached Figure 4 , select the best reaction potential, apply constant voltage to react for 60S, take samples intermittently, and use a Biowave UV-Vis spectrophotometer to determine the yield of regenerated active 1,4-NADH. Figure 5 By observing Figure 4 and 5 It can be found that when titanium foam is used as the working electrode, a nicotinamide adenine dinucleotide yield of more than 95% can be achieved by reacting at a voltage of -0.7V for 5 to 60 seconds.

[0032] Example 5 This embodiment uses foamed metal as a base electrode and adds carbon nanoparticles to the reaction solution to form a foamed metal-carbon nanoelectrode (to avoid conflict with the homemade composite metal-carbon electrode, it is called Ti foam (GN)). Specifically, this embodiment discloses a method for preparing nicotinamide adenine dinucleotide using a foamed metal-carbon electrode, which specifically includes the following steps: using PBS buffer (0.1M, pH7.5) to prepare 5 mmol / L NAD in a volume of 100 mL + solution, after adding carbon nanoparticles, the reactor was placed on an electromagnetic stirring heater at 35 ° C and 600 r / min, the foam titanium was clamped into the working electrode of the electrochemical workstation, the glassy carbon was clamped into the auxiliary electrode of the electrochemical workstation, and the Pt|Hg(I)Hg2Cl2(s)|KCl was connected to the reference electrode. Set different voltages on the electrochemical workstation to react for 5 seconds, refer to the attached Figure 6 , select the best reaction potential, apply constant voltage to react for 60S, take samples intermittently, and use a Biowave UV-Vis spectrophotometer to determine the yield of regenerated active 1,4-NADH. Figure 7 By observing Figure 6 and 7 It can be found that when titanium foam metal is used as the working electrode and carbon nanoparticles are added to the reaction solution, the yield is as high as 99.3% under a low negative voltage of -0.7V for 3S.

[0033] Example 6 In this embodiment, metal is doped into carbon to prepare a composite metal electrode. The preparation method of the metal-carbon electrode material is as follows: carbon nanopowder, activator (KOH), ethanol and water are placed in a round-bottom flask and magnetically stirred for 24 hours to fully compound, and the water is dried in an 80°C oven. The muffle furnace used is kept at 800°C for 8 hours, cooled naturally to room temperature, washed with water, and 30mmol / L HCl is added to adjust the pH to 7.0, and dried naturally. Carbon, metal and adhesive are then compounded in a ratio of 8:1:1 for 4 hours, and evenly and thinly coated on the surface of the foam titanium and foam nickel-molybdenum electrodes with a thickness of about 0.2 mm, placed in a vacuum oven at 80°C for drying, and then sintered again in a muffle furnace at 200°C to form a metal-carbon electrode. The method for preparing nicotinamide adenine dinucleotide from a composite electrode disclosed in this embodiment specifically includes the following steps: PBS buffer (0.1M, pH7.5) is used to configure 5 mmol / L NAD with a volume of 100 mL + After adding carbon nanoparticles to the solution, the reactor was placed on an electromagnetic stirring heater at 35°C and 600r / min, the composite electrode was clamped into the working electrode of the electrochemical workstation, the glassy carbon was clamped into the auxiliary electrode of the electrochemical workstation, and Pt|Hg(I)Hg2Cl2(s)|KCl was connected to the reference electrode. Set different voltages on the electrochemical workstation to react for 5 seconds, refer to the attached Figure 6 , select the best reaction potential, apply constant voltage to react for 60S, take samples intermittently, and use a Biowave UV-Vis spectrophotometer to determine the yield of regenerated active 1,4-NADH. Figure 7 By observing Figure 6 and 7 It can be found that when the composite metal is used as the working electrode and the reaction is performed at a low negative voltage of -0.7V for 5S, the yield of nicotinamide adenine dinucleotide is as high as 99.4%.

Claims

1. A method for preparing nicotinamide adenine dinucleotide, characterized in that: Oxidized nicotinamide adenine dinucleotide (NAD + ) as raw material, a metal electrode as a working electrode, glassy carbon as an auxiliary electrode, and a Pt|Hg(I)Hg2Cl2(s)|KCl electrode as a reference electrode to electrochemically synthesize nicotinamide adenine dinucleotide; the metal electrode contains one or more metals selected from platinum, palladium, copper and titanium; the conditions for the electrochemical synthesis are: voltage -0.5~-1.8 V.

2. The method for preparing nicotinamide adenine dinucleotide according to claim 1, characterized in that: The metal electrode is palladium metal, and the conditions of the electrochemical synthesis are: voltage -1.4 V, time 3s.

3. The method for preparing nicotinamide adenine dinucleotide according to claim 1, characterized in that: The metal is foamed metal.

4. The method for preparing nicotinamide adenine dinucleotide according to claim 3, characterized in that: The metal electrode is titanium foam, and the conditions for the electrochemical synthesis are: voltage -0.7 V, time 5 to 60 s.

5. The method for preparing nicotinamide adenine dinucleotide according to claim 3, characterized in that: The metal electrode is foamed copper, and the conditions for the electrochemical synthesis are: voltage -1.1V, time 6 s.

6. The method for preparing nicotinamide adenine dinucleotide according to claim 3, characterized in that: The electrochemical synthesis reaction solution contains carbon nanoparticles.

7. The method for preparing nicotinamide adenine dinucleotide according to claim 6, characterized in that: The metal electrode is titanium foam; the conditions of the electrochemical synthesis are: voltage -0.7 V, time 3s.

8. The method for preparing nicotinamide adenine dinucleotide according to claim 1, characterized in that: The metal electrode is prepared by calcining the metal and carbon; the conditions of the electrochemical synthesis are: voltage -0.7 V, time 5s.

9. The method for preparing nicotinamide adenine dinucleotide according to claim 1, characterized in that: The electrochemical synthesis conditions also include: temperature 35° C., pressure 1.01 MPa, and rotation speed 600 rpm.

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