Separation and purification method for synthesizing NADP through whole enzyme method

Abstract

The invention discloses a separation and purification method for synthesizing NADP (Nicotinamide Adenine Dinucleotide) by a whole enzyme method, which comprises the steps of membrane impurity removal, decoloration and pigment removal, electrodialysis desalination, nanofiltration, concentration, alcohol precipitation and the like, by adopting the purification method, a pure NAD product with the purity of more than 99% can be prepared from a crude NAD product solution prepared by a biological catalysis method, and the yield is about 85%. The purification method has the advantages of simple and fast operation, no need of use of a large amount of toxic and harmful reagents, small environmental pollution and low purification cost, and is suitable for large-scale and large-scale industrial separation and purification of NADP products from biological enzyme reaction liquid and biological fermentation liquid.

Description

Technical Field

[0001] The invention belongs to the field of biochemistry, and in particular relates to a method for separating and purifying a nicotinamide adenine dinucleotide phosphate product prepared by a biocatalytic method. Background Art

[0002] NADP is widely present in organisms but its content is extremely low. At present, there are many methods for preparing nicotinamide adenine dinucleotide phosphate (NADP), including chemical synthesis, bioenzyme catalysis and yeast fermentation extraction. The chemical synthesis method is inherently polluted and harsh in chemical industry, and the yield is not high, and the synthesis cost is high. The traditional biological method is to use fermentation or other microbial culture technology, and obtain NADP by separating and extracting yeast or other microorganisms. Although this process is very mature, it consumes huge raw materials, has high labor intensity, high energy consumption, limited output, high production cost and high product price. Therefore, this method has great limitations in industrial preparation scale. The bioenzyme catalysis of NADP is mainly obtained by catalyzing the phosphorylation of NAD under the action of NAD kinase, using ATP or polyphosphate (poly (P)) derivatives as phosphate donors. NAD kinase is divided into two categories, one is ATP NAD kinase, which uses ATP as a phosphoryl donor to catalyze the phosphorylation of NAD, and the other is poly (P) / ATP NAD kinase, which uses poly (P) and ATP as phosphoryl donors to catalyze the phosphorylation of NAD. Because of its inherent advantages such as high efficiency and green environmental protection, enzyme catalysis has gradually become the mainstream method for industrial preparation of NADP.

[0003] However, the existing technology still has the problems of low yield of NADP synthesis, high production cost, complicated synthesis steps, long production cycle, etc., and the traditional purification process of bio-enzyme catalysis mostly adopts ion exchange resin purification and other means. Due to the influence of the synthesis process of coenzyme II, the crude product generally contains more phosphate, and the phosphate is not easy to remove by ion exchange purification method, and it is also difficult to remove it completely during the nanofiltration concentration process. Therefore, it is necessary to develop a preparation method of NADP with simple process, short cycle, low cost and high product yield, which is easy to realize industrialization and large-scale production. Summary of the invention

[0004] In view of the shortcomings of the prior art, the purpose of the present invention is to develop a method for preparing NADP with simple process, short cycle, low cost and high product yield, which is easy to realize industrialization and large-scale production.

[0005] The purpose of the present invention can be achieved by the following measures:

[0006] A separation and purification method for synthesizing NADP by a holoenzymatic method, the method comprising the following steps:

[0007] 1) Membrane impurity removal: The pH value of the NADP reaction solution is adjusted to 4.0-4.5 for pretreatment, and the pretreated coenzyme II solution is successively microfiltered and ultrafiltered using a membrane concentration device, and the crude product solution after impurity removal is collected;

[0008] 2) Decolorization and pigment removal: Add activated carbon to the ultrafiltration clear liquid for decolorization and pigment removal.

[0009] 3) Electrodialysis desalination: The clarified filtrate after decolorization is subjected to electrodialysis desalination.

[0010] 4) Nanofiltration concentration and alcohol precipitation: The desalted NADP solution is concentrated with a 150D nanofiltration membrane to 35%. Anhydrous ethanol 3 times the volume of the material is added to cool the NADP treatment solution to crystallize. After the crystals are precipitated, filter and collect the filtrate;

[0011] 5) Crystallization: Control the temperature of the filtrate from step 2) to 10°C, maintain the pH at 2.5-3.0, stir and grow crystals, and when the crystallization rate of NADP in the solution is above 90%, filter and collect the filter cake, and after drying, obtain a high-purity NADP product.

[0012] The separation and purification of the present invention can be applied to all subsequent treatments of the whole enzyme method for synthesizing NADP in the prior art. For example, in one scheme, the enzymatic reaction system for preparing nicotinamide adenine dinucleoside phosphate by biological enzymes is as follows: 30-50 M of NAD (nicotinamide adenine dinucleotide), 10-20 g / L of NAD kinase, 30-50 M of magnesium chloride, 50-70 mg / ml of sodium hexametaphosphate, a reaction pH of 6.5-7.5, and a reaction at 30-36° C. for 6-8 hours to obtain a NADP reaction solution. The CAS number of NAD kinase in the reaction system is 9032-66-0. Under the action of NAD kinase, magnesium chloride is added to neutralize some negative charges on the polyphosphate chain, reducing the nonspecific ionic interaction between the polyphosphate groups of the enzyme and the nucleotide, thereby promoting the transfer of phosphate. Sodium hexametaphosphate provides phosphate groups to ultimately generate NADP+. In this method, hexametaphosphate is used instead of ATP as a phosphate group donor, which greatly reduces the cost of raw materials, is simple to operate, and has a thorough reaction, making it easy to realize industrial and large-scale production.

[0013] The pH value in step 1) of the present invention can be adjusted using conventional reagents and methods in the art, such as using hydrochloric acid with a concentration of 1 mol / L for adjustment. Preferably, the pH value is adjusted to 4.5.

[0014] In step 1) of the present invention, a membrane concentration device is used to successively perform microfiltration and nanofiltration, wherein a 10NM ceramic membrane is used for microfiltration and a 10KD organic membrane is used for nanofiltration, and both membranes are commercially available.

[0015] The amount of activated carbon added in the decolorization in step 2) of the present invention can be based on the conventional method in the art, for example, 1% by volume of activated carbon is added for decolorization.

[0016] The step 3) of the present invention, electrodialysis desalination, can be carried out according to conventional procedures in the art, and the desalination is terminated when the desalination reaches 1-1.5 ms / cm.

[0017] In step 4) of the present invention, the amount of ethanol used is 2.5 to 3.5 times the volume of the concentrated solution. In a preferred embodiment, the desalted NADP solution is concentrated to 35% using a 150D nanofiltration membrane, the material is slowly added to anhydrous ethanol, cooled to 20°C, crystallized for 60 minutes, and then cooled to 10°C, the pH is maintained at 2.5 to 3.0, the crystals are stirred for 3 hours, filtered, the filter cake is collected, and a high-purity NADP product is obtained after drying.

[0018] The drying in step 4) of the present invention is carried out at 35° C. and -0.1 MPa for 5-8 hours.

[0019] The purification method of NADP provided by the present invention has the advantages of high yield and high purity of the purified product. It is verified by the examples that the purification method can be used to prepare a pure NAD product with a purity of more than 99% from a crude product solution of NAD prepared by a biocatalytic method, and the yield is about 85%. The purification method is simple and fast to operate, does not require the use of a large amount of toxic and harmful reagents, has little pollution to the environment, and has a low purification cost. It is suitable for large-scale industrial separation and purification of NADP products from bioenzyme reaction liquids and biofermentation liquids. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the crystal diagram of Example 1;

[0021] Figure 2 This is the crystal diagram of Comparative Example 1. DETAILED DESCRIPTION

[0022] The following examples are provided for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0023] Unless otherwise specified, the NADP reaction solution used for separation and purification in the following examples was prepared according to the following enzymatic reaction system: reactant NAD 40M, NAD kinase (CAS No. 9032-66-0) was added in an amount of 15 g / L, magnesium chloride was added in an amount of 40 M, sodium hexametaphosphate was added in an amount of 60 mg / ml, the reaction pH was 7.0, and the reaction was carried out at 35° C. for 7 h to obtain the NADP reaction solution. The reaction results of different batches are shown in Tables 1 and 2.

[0024] Table 1

[0025]

[0026] Table 2

[0027]

[0028]

[0029] Unless otherwise specified, the following examples all use the NADP reaction solution for subsequent separation and purification experiments.

[0030] If not otherwise specified, the 10NM ceramic membrane used in the following examples was purchased from Nanjing Jiuanyuan; the 10KD organic membrane was purchased from Nanjing Jiuanyuan's 10KD ultrafiltration, number: 230685; the 150D nanofiltration membrane was purchased from Nanjing Jiuanyuan's 150D nanofiltration membrane, number: 230277.

[0031] Example 1

[0032] S1. Membrane: The NADP reaction solution in Table 1 was pretreated by adding hydrochloric acid with a concentration of 1 mol / l to adjust the pH value to 4.0, the pretreated NADP solution was microfiltered with a 10NM ceramic membrane, and the microfiltration clear liquid was nanofiltered and concentrated with a 10KD organic membrane to collect the concentrated crude solution;

[0033] S2, impurity removal: the crude solution obtained in S1 is kept at a pH of 4.0, and 1% by volume of activated carbon is added for decolorization. After decolorization, the light transmittance of the material reaches 91.3T%;

[0034] S3, desalination: The material obtained in S2 is subjected to electrodialysis desalination. After desalination, the conductivity is reduced from 33ms / cm to 1ms / cm.

[0035] S4, secondary concentration: The desalted liquid of S3 is concentrated to 35% using a 150D nanofiltration membrane;

[0036] S5, alcohol precipitation crystallization: add hydrochloric acid to the concentrated solution obtained from S4 to adjust the pH to 2.5, take 3 times the volume of anhydrous ethanol of the concentrated solution of S3, slowly add the material into anhydrous ethanol, control the temperature at 20 degrees, stir and crystallize for about 1 hour, the crystallization rate is about 90%, continue to cool to 10 degrees, grow crystals for 3 hours, filter and collect the filter cake, dry it at 35°C and -0.1Mpa for 5-8 hours to obtain a high-purity NADP product. The results are shown in Table 3 and Figure 1 .

[0037] Example 2

[0038] S1. Membrane: The NADP reaction solution in Table 1 was pretreated by adding hydrochloric acid with a concentration of 1 mol / l to adjust the pH value to 4.5, the pretreated NADP solution was microfiltered with a 10NM ceramic membrane, and the microfiltration clear liquid was nanofiltered and concentrated with a 10KD organic membrane to collect the concentrated crude solution;

[0039] S2, impurity removal: the crude product solution obtained in S1 is kept at a pH of 4.5, and 1% by volume of activated carbon is added for decolorization. After decolorization, the light transmittance of the material reaches 90.9T%;

[0040] S3, desalination: The material obtained in S2 is subjected to electrodialysis desalination. After desalination, the conductivity is reduced from 32ms / cm to 1.2ms / cm.

[0041] S4, secondary concentration: The desalted liquid of S3 is concentrated to 35% using a 150D nanofiltration membrane;

[0042] S5, alcohol precipitation crystallization: add hydrochloric acid to the concentrated solution obtained from S4 to adjust the pH to 2.5, take 3 times the volume of anhydrous ethanol of the concentrated solution of S3, slowly add the material into anhydrous ethanol, control the temperature at 20 degrees, stir and crystallize for about 1 hour, the crystallization rate is about 90%, continue to cool to 10 degrees, incubate the crystal for 3 hours, filter and collect the filter cake, dry it at 35°C and -0.1Mpa for 5-8 hours to obtain a high-purity NADP product. The results are shown in Table 3.

[0043] Table 3

[0044]

[0045] Example 3

[0046] S1. Membrane: The NADP reaction solution in Table 2 is pretreated by adding hydrochloric acid with a concentration of 1 mol / l to adjust the pH value to 4.5, the pretreated NADP solution is microfiltered with a 10NM ceramic membrane, and the microfiltration clear liquid is nanofiltered and concentrated with a 10KD organic membrane to collect the concentrated crude solution;

[0047] S2, impurity removal: the crude product solution obtained in S1 is kept at a pH of 4.5, and 1% by volume of activated carbon is added for decolorization. After decolorization, the light transmittance of the material reaches 91.9T%;

[0048] S3, desalination: The material obtained in S2 is subjected to electrodialysis desalination. After desalination, the conductivity is reduced from 32ms / cm to 1.2ms / cm.

[0049] S4, secondary concentration: The desalted liquid of S3 is concentrated to 35% using a 150D nanofiltration membrane;

[0050] S5, alcohol precipitation crystallization: add hydrochloric acid to the concentrated solution obtained from S4 to adjust the pH to 2.5, take 3 times the volume of anhydrous ethanol of the concentrated solution of S3, slowly add the material into anhydrous ethanol, control the temperature at 20 degrees, stir and crystallize for about 1 hour, the crystallization rate is about 90%, continue to cool to 10 degrees, incubate the crystal for 3 hours, filter and collect the filter cake, dry it at 35°C and -0.1Mpa for 5-8 hours to obtain a high-purity NADP product. The results are shown in Table 4.

[0051] Example 4

[0052] S1. Membrane: The NADP reaction solution in Table 2 is pretreated by adding hydrochloric acid with a concentration of 1 mol / l to adjust the pH value to 4.5, the pretreated NADP solution is microfiltered with a 10NM ceramic membrane, and the microfiltration clear liquid is nanofiltered and concentrated with a 10KD organic membrane to collect the concentrated crude solution;

[0053] S2, impurity removal: the crude solution obtained in S1 is kept at a pH of 4.5, and 1% by volume of activated carbon is added for decolorization. After decolorization, the light transmittance of the material reaches 92.9T%;

[0054] S3, desalination: The material obtained in S2 is subjected to electrodialysis desalination. After desalination, the conductivity is reduced from 32ms / cm to 1.2ms / cm.

[0055] S4, secondary concentration: The desalted liquid of S3 is concentrated to 35% using a 150D nanofiltration membrane;

[0056] S5, alcohol precipitation crystallization: add hydrochloric acid to the concentrated solution obtained from S4 to adjust the pH to 2.5, take 3 times the volume of anhydrous ethanol of the concentrated solution of S3, slowly add the material into anhydrous ethanol, control the temperature at 20 degrees, stir and crystallize for about 1 hour, the crystallization rate is about 90%, continue to cool to 10 degrees, incubate the crystal for 3 hours, filter and collect the filter cake, dry it at 35°C and -0.1Mpa for 5-8 hours to obtain a high-purity NADP product. The results are shown in Table 4.

[0057] Table 4

[0058]

[0059] Comparative Example 1

[0060] The difference between this comparative example and Example 1 is that in step S5, the material is added back to ethanol instead of being added to the material in the forward direction: hydrochloric acid is added to the concentrated solution obtained in S4 to adjust the pH to 2.5, and anhydrous ethanol with a volume of 3 times that of the concentrated solution in S3 is taken, and the anhydrous ethanol is slowly added to the material, the temperature is controlled at 20 degrees, and the material is stirred. The material gradually turns milky white. After about 30 minutes, the ethanol addition is completed, and an oily lump is formed at the bottom of the material, and crystallization fails ( Figure 2 ).

Claims

1. A separation and purification method for synthesizing NADP by a holoenzymatic method, characterized in that: The method comprises the following steps: 1) Membrane removal: The pH value of the NADP reaction solution is adjusted to 4.0-4.5, and microfiltration and nanofiltration are performed successively using a membrane concentration device to collect the crude product solution after impurities are removed; 2) Decolorization and pigment removal: Add activated carbon to the crude product solution to decolorize and remove the pigment; 3) Electrodialysis desalination: The clarified filtrate after decolorization is subjected to electrodialysis desalination; 4) Nanofiltration concentration and alcohol precipitation: The desalted NADP solution is concentrated to 30-37% using a 150D nanofiltration membrane to form a concentrated solution, which is slowly added to anhydrous ethanol, cooled to 18-22°C, and crystallized for 50-60 minutes. The temperature is further cooled to 8-10°C, the pH is maintained at 2.5-3.0, and the crystals are stirred for 2-3 hours. The filter cake is collected and dried to obtain a high-purity NADP product.

2. The method according to claim 1, characterized in that The NADP reaction solution in step 1) is obtained by the following method: the reactant NAD is 30-50M, the addition amount of NAD kinase is 10-20g / L, the addition amount of magnesium chloride is 30-50M, the addition amount of sodium hexametaphosphate is 50-70mg / ml, the reaction pH is 6.5-7.5, and the reaction is carried out at 30-36°C for 6-8h to obtain the NADP reaction solution.

3. The method according to claim 1, characterized in that Step 1) The pH value was adjusted to 4.

5.

4. The method according to claim 1, characterized in that: The microfiltration uses 10NM ceramic membrane, and the nanofiltration uses 10KD organic membrane.

5. The method according to claim 1, characterized in that Step 3) The electrodialysis desalination is terminated when the concentration reaches 1-1.5 ms / cm.

6. The method according to claim 1, characterized in that The amount of ethanol used in step 4) is 2.5 to 3.5 times the volume of the concentrated solution.

7. The method according to claim 1, characterized in that Step 4) The desalted NADP solution is concentrated to 35% using a 150D nanofiltration membrane, the material is slowly added to anhydrous ethanol, cooled to 20°C, crystallized for 60 minutes, and then cooled to 10°C, the pH is maintained at 2.5-3.0, and the crystals are stirred for 3 hours, filtered, and the filter cake is collected and dried to obtain a high-purity NADP product.

8. The method according to claim 1, characterized in that The drying in step 4) is carried out at 35° C. and −0.1 MPa for 5-8 h.

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