Process for preparing NAD < + > through enzymatic catalysis

Abstract

The invention provides a method for preparing NAD < + > by enzymatic catalysis, which comprises the following steps: adding nicotinamide mononucleotide, disodium adenosine triphosphate and magnesium chloride hexahydrate into an enzymatic reaction system, and carrying out catalytic reaction by taking nicotinamide mononucleotide adenylate transferase and pyrophosphatase as catalysts to obtain nicotinamide adenine dinucleotide. According to the method, enzymatic catalysis is adopted, an enzymatic catalytic reaction system and process of NAD < + > are established, NAD < + > is obtained through a catalytic reaction of NMN and adenosine disodium triphosphate, the catalytic reaction is set according to optimized reaction conditions, and the yield can reach 99% or above.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme engineering, and in particular to a preparation technology for synthesizing nicotinamide adenine dinucleotide by enzymatic catalysis. Background Art

[0002] Nicotinamide adenine dinucleotide (NAD+, coenzyme I) and its phosphorylated and reduced forms (NADP+, NADH and NADPH) are coenzymes of oxidoreductases and participate in more than 400 enzyme-catalyzed reactions. They are important coenzymes in living organisms and play a role in transferring hydrogen anions and electrons in enzymatic reactions. They play an important role in life processes such as redox, gene expression, energy metabolism, and regulation of cell life cycle and disease. In addition to being a cofactor of oxidoreductases with a single enzymatic activity, they also serve as substrates for a variety of proteins in various signal transduction mechanisms. The biocatalytic reactions in which this type of coenzyme participates have the characteristics of enzyme catalysis, namely, high efficiency, strong selectivity, and high yield.

[0003] The synthesis methods of NAD+ are mainly divided into chemical synthesis, yeast extraction and enzymatic preparation. The early chemical synthesis synthesized NAD+ from AMP and β-NMN, but this method has the disadvantages of long reaction time, complex process, high cost and low yield, so it is limited in industrial production. At present, a large number of NAD+ products in industry are mainly extracted and separated from yeast. The process is mature, but the high energy consumption and high cost limit the production of NAD+ and the development and application of subsequent products. Compared with the chemical method, the preparation of NAD+ by biocatalysis has the advantages of simple process conditions, green environmental protection, and short time. Therefore, it is necessary to develop a low-cost, cost-controlled, and efficient biosynthetic route. Summary of the invention

[0004] In order to solve the above-mentioned technical problems existing in the prior art, the present invention adopts enzymatic catalysis to establish an enzymatic catalytic reaction system and process of NAD+, and obtains NAD+ by catalytic reaction of NMN and disodium ATP. The catalytic reaction is set according to the optimized reaction conditions, and the yield can reach more than 99%.

[0005] In the present invention, nicotinamide mononucleotide adenylate transferase and pyrophosphatase are used in combination as catalysts, and nicotinamide mononucleotide adenylate transferase and pyrophosphatase can play a synergistic catalytic role, so that the conversion rate of NAD+ synthesis can be improved. The enzyme is in the form of lyophilized powder, bacterial liquid or bacterial mud. In a specific embodiment, the present invention uses a bacterial mud form.

[0006] A method for preparing NAD+ by using enzymatic catalysis, wherein nicotinamide mononucleotide, disodium adenosine triphosphate, and magnesium chloride hexahydrate are added to an enzymatic reaction system, and a catalytic reaction is carried out using nicotinamide mononucleotide adenylate transferase and pyrophosphatase as catalysts to obtain the nicotinamide adenine dinucleotide.

[0007] Furthermore, in the enzymatic reaction system, the molar ratio of nicotinamide mononucleotide, adenosine triphosphate disodium and magnesium chloride hexahydrate is (0.15-0.2):(0.15-0.25):(0.25-0.4), and preferably in the enzymatic reaction system, the molar ratio of nicotinamide mononucleotide, adenosine triphosphate disodium and magnesium chloride hexahydrate is 0.15:0.18:0.3, 0.15:0.2:0.3, 0.2:0.22:0.3, 0.2:0.23:0.4.

[0008] Furthermore, the catalytic reaction is carried out under stirring at a stirring speed of 100-300 rpm; preferably, the stirring speed is 120 rpm, 150 rpm, 200 rpm, 250 rpm or 280 rpm.

[0009] Furthermore, the temperature of the catalytic reaction is 30-40°C; preferably, the temperature of the catalytic reaction is 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C.

[0010] Furthermore, the pH of the catalytic reaction is 6.0-7.5; preferably, the pH of the catalytic reaction is pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4 or pH 7.5.

[0011] Furthermore, the bacterial concentration of the catalytic reaction is 8-20 g / L; preferably, the bacterial concentration of the catalytic reaction is 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L or 20 g / L.

[0012] The beneficial effects of the present invention are:

[0013] (1) The present invention provides a process for enzymatically synthesizing nicotinamide adenine dinucleotide, which is simple and can achieve the effect of efficiently synthesizing NAD+, reduce production costs, and reduce the difficulty of subsequent reaction processing.

[0014] (2) The preparation process of the present invention, through the enzymatic synthesis of nicotinamide adenine dinucleotide, can increase the generation rate of the reaction product NAD+, and the product generation rate is ≥99%.

[0015] (3) The enzymatic reaction system of the present invention is simple, easy to operate, has a stable reaction, a high conversion rate, and is easy to industrialize and mass-produce. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : The initial sample solution of the sample prepared in Example 3, the liquid chromatogram when there is no enzyme solution;

[0017] Figure 2 : Liquid chromatogram of the sample prepared in Example 3 at 0h of the catalytic reaction;

[0018] Figure 3 : Liquid chromatogram of the sample prepared in Example 3 at the 1st hour of the catalytic reaction;

[0019] Figure 4 : Liquid chromatogram of the sample prepared in Example 3 at the 2nd hour of the catalytic reaction;

[0020] Figure 5 : Liquid chromatogram of the sample prepared in Example 3 at the 3rd hour of the catalytic reaction;

[0021] Figure 6 : Liquid chromatogram of the sample prepared in Example 3 at 4h of the catalytic reaction. DETAILED DESCRIPTION

[0022] The present invention is further explained below with reference to the examples, but the examples do not limit the present invention in any form.

[0023] Example 1 Preparation of crude enzyme solution

[0024] Take out the nicotinamide mononucleotide adenylate transferase and pyrophosphatase bacterial slurry stored in the refrigerator, weigh an appropriate amount of bacteria to dissolve, use a homogenizer to break the bacteria, repeat the homogenization three times, and the final fixed volume broken bacteria concentration is 200g / L.

[0025] Example 2 Laboratory Preparation of NAD+

[0026] In a 1L reaction system, the stirring speed was set to 250rpm and the temperature was set to 35°C. 50.13g of nicotinamide mononucleotide (0.15mol), 99.21g of disodium adenosine triphosphate (0.18mol) and 60.99g of magnesium chloride hexahydrate (0.3mol) were weighed in advance and dissolved in deionized water. After dissolution and mixing, the pH was adjusted to 6.3, and the crushed bacterial sludge was added for reaction (the concentration of the bacterial sludge was 8g / L). The total volume was 1000mL. The catalytic reaction was carried out under stirring at a stirring speed of 120rpm. The pH of the catalytic reaction system was maintained at 6.5, and the temperature of the catalytic reaction was 35°C. After 4 hours of reaction, the NMN residue was detected to be ≤1.0g / L, the NAD+ production was ≧99g / L, and the yield was 99.5%.

[0027] Example 3 Amplification of the reaction to prepare NAD+

[0028] Enzymatic synthesis of NAD+ amplification reaction, set the stirring speed to 120rpm, the temperature to 35℃, use a 20L reactor as the reaction vessel, weigh 802.1g (2.4mol) of finished nicotinamide mononucleotide, 1587.3g (2.88mol) of disodium adenosine triphosphate, and 975.8g (4.8mol) of magnesium chloride hexahydrate, dissolve them in deionized water, adjust the pH to 6.3, add crushed bacterial sludge to react (the concentration of bacterial sludge is 8g / L), maintain pH 6.5, and after 4 hours of reaction, the residual NMN is detected to be 0.49-2.89g / L, the NAD+ production is ≧97g / L, and the yield is 99%. Some experimental results are shown in Table 1 and Figure 1 , 2 , 3, 4, 5, and 6.

[0029] Table 1:

[0030]

[0031] Table 1 and Figure 1 , 2 The results of 3, 4, 5, and 6 show that a large amount of NAD+ is generated immediately after the enzyme solution is added. After 1 hour of reaction, the NAD+ generation is ≧90g / L, and the NMN residue is ≤1.7g / L. In the following 3 hours, the output remains at 97g / L, and the NMN residue is ≤1.0g / L. The enzyme-catalyzed synthesis process of the present invention can efficiently synthesize NAD+, with stable reaction and high conversion rate.

[0032] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing NAD+ by enzymatic catalysis, characterized in that: Nicotinamide mononucleotide, disodium adenosine triphosphate and magnesium chloride hexahydrate are added to the enzymatic reaction system, and nicotinamide mononucleotide adenylate transferase and pyrophosphatase are used as catalysts to carry out a catalytic reaction to obtain the nicotinamide adenine dinucleotide.

2. The method according to claim 1, characterized in that In the enzymatic reaction system, the molar ratio of nicotinamide mononucleotide, adenosine triphosphate disodium and magnesium chloride hexahydrate is (0.15-0.2):(0.15-0.25):(0.25-0.4). Preferably, in the enzymatic reaction system, the molar ratio of nicotinamide mononucleotide, adenosine triphosphate disodium and magnesium chloride hexahydrate is 0.15:0.18:0.3, 0.15:0.2:0.3, 0.2:0.22:0.3, 0.2:0.23:0.

4.

3. The method according to claim 1, characterized in that The catalytic reaction is carried out under stirring at a stirring speed of 100-300 rpm; preferably, the stirring speed is 120 rpm, 150 rpm, 200 rpm, 250 rpm or 280 rpm.

4. The method according to claim 1, characterized in that The temperature of the catalytic reaction is 30-40°C; preferably, the temperature of the catalytic reaction is 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C.

5. The method according to claim 1, characterized in that The pH of the catalytic reaction is 6.0-7.5; preferably, the pH of the catalytic reaction is pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4 or pH 7.

5.

6. The method according to claim 1, characterized in that The bacterial concentration of the catalytic reaction is 8-20 g / L; preferably, the bacterial concentration of the catalytic reaction is 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L or 20 g / L.

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