阅读说明：本技术 一种应用生物酶法制备nadp的方法 (Method for preparing NADP by applying biological enzyme method ) 是由 王康林 汪晓东 王磊 金永红 于 2021-07-19 设计创作，主要内容包括：本发明涉及生物制药领域,具体涉及应用生物酶制备烟酰胺腺嘌呤二核苷磷酸的方法。所述方法包括建立应用生物酶制备烟酰胺腺嘌呤二核苷磷酸的酶促反应体系,所述生物酶选自NAD激酶,所述NAD激酶的CAS号为9032-66-0；所述酶促反应体系还包括NAD、偏磷酸盐、pH调节剂、以及Mg～(2+)和/或Co～(2+)。所述方法还包括取偏磷酸盐、NAD激酶、pH调节剂、以及含Mg～(2+)和/或Co～(2+)的物质定溶,通过连续流加底物的方式进行反应。本发明要解决的技术问题在于克服现有技术中NADP合成的收率低、生产成本高,以及合成步骤繁琐、生产周期长的问题,提供一种生物酶高效制备NADP的生产方法。其工艺简单、周期短、成本低以及产物收率高,易实现产业化、规模化生产。(The invention relates to the field of biological pharmacy, in particular to a method for preparing nicotinamide adenine dinucleotide phosphate by using biological enzyme. The method comprises the steps of establishing an enzymatic reaction system for preparing nicotinamide adenine dinucleotide phosphate by using a biological enzyme, wherein the biological enzyme is selected from NAD kinase, and the CAS number of the NAD kinase is 9032-66-0; the enzymatic reaction system further comprises NAD, metaphosphate, pH regulator, and Mg 2+ And/or Co 2+ . The method further comprises taking metaphosphate, NAD kinase, pH regulator, and Mg 2+ And/or Co 2+ The substance (2) is dissolved and reacted by continuously feeding the substrate. The invention aims to solve the technical problems of low yield, high production cost, complex synthesis steps and long production period of NADP synthesis in the prior art, and provides a production method for efficiently preparing NADP by using biological enzyme. The method has the advantages of simple process, short period, low cost, high product yield and easy realization of productionIndustrialized and large-scale production.)

1. A method for preparing nicotinamide adenine dinucleotide phosphate by using biological enzyme is characterized in that: the method comprises the steps of establishing an enzymatic reaction system for preparing nicotinamide adenine dinucleotide phosphate by using a biological enzyme, wherein the biological enzyme is selected from NAD kinase, and the CAS number of the NAD kinase is 9032-66-0;

the enzymatic reaction system further comprises NAD, metaphosphate, pH regulator, and Mg²⁺And/or Co²⁺。

2. The method of claim 1, wherein Mg in said enzymatic reaction system²⁺0.01-0.1mol/L and/or Co²⁺0.01-0.1 mol/L; preferably, Mg in the enzymatic reaction system²⁺Is 0.05mol/L and/or Co²⁺Is 0.03 mol/L.

3. The method according to claim 1, wherein the enzymatic reaction system contains NAD 5-15%, and/or metaphosphate 10-30%, and/or the enzyme base ratio is 5:1-50:1, and/or the pH of the enzymatic reaction system is adjusted to 6.0-8.0 by a pH adjusting agent.

4. The method of claim 1, wherein the metaphosphate comprises sodium trimetaphosphate, sodium tetrametaphosphate, sodium hexametaphosphate, potassium trimetaphosphate, potassium tetrametaphosphate, potassium hexametaphosphate, and/or the pH adjuster comprises PBS buffer, sodium acetate-acetate, borate; and/or said Mg²⁺Selected from MgSO₄·7H₂O, the Co²⁺Selected from CoCl₂。

5. The method of claim 1, wherein the enzymatic reaction system comprises NAD 10%, and/or metaphosphate 20%, and/or enzyme base ratio 30:1, and/or pH adjusting agent to adjust pH of the composition to 7.0.

6. The method of any one of claims 1 to 5, wherein the metaphosphate, NAD kinase, pH adjusting agent, and Mg are taken²⁺And/or Co²⁺The substance (2) is dissolved and reacted by continuously feeding the substrate.

7. The method of claim 6, wherein the temperature of the reaction is between 28 ℃ and 43 ℃; preferably 37 deg.c.

8. The method as claimed in claim 6, wherein the rotation speed of the solution in the reaction is 100-1000 r/min; preferably 250 r/min.

9. The process according to claim 6, wherein the reaction time is from 0.5 to 10 hours.

10. The method of claim 9, wherein the reaction time is 3 hours.

Technical Field

The invention belongs to the field of biological pharmacy, and particularly relates to a method for preparing nicotinamide adenine dinucleotide phosphate by using a biological enzyme preparation.

Background

Nicotinamide Adenine Dinucleotide Phosphate (NADP), also known as pyridine triphosphate, is an oxidized coenzyme widely involved in redox metabolism and other biochemical reactions of organisms, and is used as a hydrogen transporter in the process of biological oxidation to participate in the synthesis of lipids, nucleotides, fatty acids and the like in the form of reduced NADPH, so that the Nicotinamide Adenine Dinucleotide Phosphate (NADP) is an essential coenzyme in organisms. Research shows that the oxidized coenzyme II can promote metabolism, energy metabolism, resist cell senility and resist oxidation.

The existing NADP synthesis method can be divided into a chemical method, a biological fermentation method and a biological enzyme method, wherein the chemical method takes nicotinamide as a raw material and synthesizes the NADP through multi-step reaction, and the chemical method has the defects of long reaction route, harsh reaction conditions, poor selectivity, easy generation of byproducts, low product purity, low yield, high cost and the like, needs expensive reagents, and causes environmental pollution due to the use of a large amount of organic solvents. Therefore, the process route is not suitable for industrial mass production. The conventional biological method is to use fermentation or other microorganism culture techniques and to isolate and extract NADP from yeast or other microorganisms. Although the process is mature, the process has the disadvantages of huge raw material consumption, high labor intensity, high energy consumption, limited yield, high production cost and high product price, and limits the wide application of oxidized coenzyme II (NADP). In the research of the NADP synthesized by the biological enzyme method, the process has the following defects: firstly, the raw material synthesis yield is low, the scale-up production is not easy, and the raw material source is limited; secondly, the enzyme catalytic synthesis of NADP can only obtain high conversion rate in gram-level range, and has long reaction time and low productivity; thirdly, the production of NADP needs to be carried out in the presence of ATP, which is expensive, thus increasing the production cost of NADP.

The enzyme is a specific high-efficiency biocatalyst, and most of the enzyme is protein produced by living cells. The catalytic condition of the enzyme is mild, and the enzyme can be carried out at normal temperature and normal pressure. The enzyme-catalyzed reaction, referred to as an enzymatic reaction, is 103-107 times faster than the corresponding uncatalyzed reaction. Enzymatic reaction kinetics is abbreviated as enzyme kinetics, and the relationship between the speed of an enzymatic reaction and the concentration of a substrate (i.e., a reactant) and other factors is mainly studied. The choice of temperature, pH, enzyme concentration, substrate concentration, inhibitors, activators, etc. in the enzymatic reaction can have a significant impact on the reaction outcome and efficiency. Therefore, the selection of suitable biological enzymatic reaction conditions is of crucial importance.

Disclosure of Invention

The invention aims to solve the technical problems of low yield, high production cost, complex synthesis steps and long production period of NADP synthesis in the prior art, and provides a production method for efficiently preparing NADP by using biological enzyme. The method has the advantages of simple process, short period, low cost, high product yield and easy realization of industrialized and large-scale production.

The purpose of the invention is realized as follows:

the invention provides a method for preparing nicotinamide adenine dinucleotide phosphate by applying biological enzyme,

the method comprises the steps of establishing an enzymatic reaction system for preparing nicotinamide adenine dinucleotide phosphate by using a biological enzyme, wherein the biological enzyme is selected from NAD kinase, and the CAS number of the NAD kinase is 9032-66-0; the enzymatic reaction system further comprises NAD, metaphosphate, pH regulator, and Mg²⁺And/or Co²⁺。

Preferably, Mg in the enzymatic reaction system²⁺0.01-0.1mol/L and/or Co²⁺0.01-0.1 mol/L; preferably, Mg in the enzymatic reaction system²⁺Is 0.05mol/L and/or Co²⁺Is 0.03 mol/L.

Preferably, the enzymatic reaction system contains NAD 5-15% and/or metaphosphate 10-30% by mass fraction, and/or the enzyme base ratio is 5:1-50:1, and/or the pH regulator regulates the pH of the enzymatic reaction system to 6.0-8.0.

Preferably, the metaphosphate comprises sodium trimetaphosphate, sodium tetrametaphosphate, sodium hexametaphosphate, potassium trimetaphosphate, potassium tetrametaphosphate, potassium hexametaphosphate, and/or the pH adjuster comprises PBS buffer, acetic acid-sodium acetate, borate; and/or said Mg²⁺Selected from MgSO₄·7H₂O, the Co²⁺Is selected fromCoCl₂。

Preferably, the enzymatic reaction system contains NAD 10% and/or metaphosphate 20% by mass fraction and/or enzyme base ratio 30:1 and/or pH regulator to adjust pH of the composition to 7.0.

The method specifically comprises the following steps: taking metaphosphate, NAD kinase, pH regulator and Mg²⁺And/or Co²⁺The substance (2) is dissolved and reacted by continuously feeding the substrate.

Preferably, the temperature of the reaction is 28-43 ℃; preferably 37 deg.c.

Preferably, the rotation speed of the solution in the reaction is 100-1000 r/min; preferably 250 r/min.

Preferably, the reaction time is 0.5-10 h; preferably 3 h; preferably, the reaction time is 3 h.

According to the invention, through the research on the enzymatic mechanism of the NAD kinase, the enzyme activator with appropriate concentration is screened and added, the activity of the enzyme is activated, and simultaneously, the enzyme plays a role of bridging when being combined with a substrate, so that the conversion rate of the nicotinamide adenine dinucleotide phosphate is greatly improved. In addition, metaphosphate is used as a phosphate group donor instead of ATP, so that the cost of raw materials is greatly reduced, the operation is simple, the reaction is thorough, and the industrial and large-scale production is easy to realize.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 Effect of different activators and concentrations on NAD kinase Activity

FIG. 2 Effect of different NAD addition concentrations on enzymatic reaction conversion

FIG. 3 Effect of different enzyme base ratios on the conversion ratio of enzymatic reactions

FIG. 4 Effect of different reaction temperatures on the conversion ratio of the enzymatic reaction

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The technical scheme of the invention is as follows:

a method for efficiently preparing nicotinamide adenine dinucleotide phosphate by using a biological enzyme preparation. Selects commercial nicotinamide adenine dinucleotide kinase (NAD kinase, CAS: 9032-66-0, Beijing Baiolai Paco technologies Co., Ltd.), and obtains the method for efficiently preparing nicotinamide adenine dinucleotide phosphate by screening and adding an enzyme activator, taking nicotinamide adenine dinucleotide and metaphosphate as raw materials, and optimizing enzymatic reaction conditions such as substrate concentration, enzyme substrate ratio, activator addition amount, pH, temperature and the like on the basis of mechanism research.

Wherein, the enzyme activity of the NAD kinase is measured: (1) assay reaction (100 mL): 25mmol/L substrate NAD, 110mmol/L sodium trimetaphosphate, 0.2mol/L PBS buffer with pH7.0, and 5g NAD kinase. (2) Shaking the prepared solution, stirring at 37 deg.C at 250r/min, sampling every 1 hr, measuring NADP peak area at 280nm by HPLC, and continuously measuring for 10 hr. The peak area is plotted against time and the initial linear portion of the reaction is taken to calculate the Δ S value. And calculating the enzyme activity according to a formula. The enzyme activity unit (U) is defined as: the amount of enzyme catalyzing the production of 1. mu. mol NADP per hour at 37 ℃ was one unit. The calculation formula is as follows:

in the formula: delta S is the peak area variation value of 280nmNADP, S is the corresponding peak area of 1g/mL of NADP standard, V is the enzymatic reaction volume (mL),743 is the molecular weight of NADP, and m is the amount (g) of NAD kinase added into the system.

Content method of HPLC detection NADP: c18 HPLC column, 150 mm. times.4.6 mm; mobile phase: 25mmol/LpH ═ 7.0Tris-HAC, methanol; an ultraviolet detector with the wavelength of 280nm, the column temperature of 30 ℃, the flow rate of 0.8mL/min and the sample injection amount of 5 mu L.

Example 1

This example illustrates the effect of enzyme activator species on the enzymatic production of nicotinamide adenine dinucleotide phosphate by adding different concentrations of Fe to the enzymatic activity assay system for NAD kinase³⁺、Mg²⁺、Mn²⁺、Ca²⁺、Zn²⁺、Co²⁺And (3) metal ion inorganic salt. The relative enzyme activity was used to determine the type and concentration of metal ions, and the results are shown in FIG. 1. The experimental result shows that Mg is added²⁺Or Co²⁺Has activating effect on NAD kinase, wherein Co²⁺Or Mg²⁺The best activation was obtained when the addition was 0.03mol/L and 0.05mol/L, respectively.

Example 2

This example illustrates the effect of different substrate concentrations on the production of nicotinamide adenine dinucleotide phosphate by an enzymatic reaction, under otherwise identical conditions, specifically: sodium trimetaphosphate 20%, enzyme base ratio 15: 1, Co²⁺And Mg²⁺0.03mol/L and 0.05mol/L, respectively, and PBS buffer (pH 7.0) were added to the reaction solution to carry out a reaction at 35 ℃ and 250r/min for 3 hours.

NAD was added at concentrations of 5%, 10%, 15%, 20% and 25%, respectively. The experimental result shows that when the concentration of the substrate is 10%, the conversion rate of the enzymatic reaction to produce nicotinamide adenine dinucleotide phosphate is highest.

Example 3

This example illustrates the effect of different enzyme substrates on the production of nicotinamide adenine dinucleotide phosphate by an enzymatic reaction, and similar to example 2, enzyme substrate ratios of 10:1, 20:1, 30:1, 40:1, 50:1 were examined, respectively, under otherwise identical conditions for the enzymatic reaction. The experimental result shows that when the enzyme substrate ratio is 30:1, the conversion rate of nicotinamide adenine dinucleotide phosphate produced by the enzymatic reaction is the highest. The enzyme substrate ratio refers to enzyme activity (U): NAD (g).

Example 4

This example illustrates the effect of different reaction temperatures on the production of nicotinamide adenine dinucleotide phosphate by the enzymatic reaction, and similarly to example 2, the reaction temperatures were examined at 28 ℃, 31 ℃, 34 ℃, 37 ℃, 40 ℃ and 43 ℃ respectively, under otherwise identical conditions for the enzymatic reaction. The experimental result shows that when the reaction temperature is 37 ℃, the conversion rate of nicotinamide adenine dinucleotide phosphate produced by the enzymatic reaction is the highest.

Example 5

50g of NAD, 100g of sodium trimetaphosphate, 24g of NAD kinase and MgSO₄·7H₂O 0.025mol，CoCl₂0.015mol, PBS buffer solution (pH 7.0) is dissolved to 500mL, reaction is carried out for 3h at the temperature of 37 ℃ and the rotating speed of 250r/min, the concentration of nicotinamide adenine dinucleotide phosphate in the system reaches 85g/L, and the conversion rate is 85%.

Example 6

Taking 150g NAD, 300g sodium trimetaphosphate, 72g NAD kinase and MgSO₄·7H₂O 0.075mol，CoCl₂0.045mol, PBS buffer solution (pH 7.0) is dissolved to 1500mL, reaction is carried out for 3h at the temperature of 37 ℃ and the rotation speed of 250r/min, the concentration of nicotinamide adenine dinucleotide phosphate in the system reaches 83g/L, and the conversion rate is about 83%.

Example 7

200g of NAD, 400g of sodium trimetaphosphate, 96g of NAD kinase and MgSO₄·7H₂O 0.1mol，CoCl₂0.06mol, dissolving in PBS buffer solution (pH 7.0) to 2000mL, reacting at 37 deg.C and 250r/min for 3h, wherein the concentration of nicotinamide adenine dinucleotide phosphate in the system reaches 70g/L, and the conversion rate is only about 70%.

Example 8

Sodium trimetaphosphate 400g, NAD kinase 96g, MgSO₄·7H₂O 0.1mol，CoCl₂0.06mol, dissolving PBS buffer solution (pH 7.0) to 2000mL, adding substrate into the reaction system by a continuous flow adding mode, adding NAD with the addition amount of 200g, reacting for 3h at the temperature of 37 ℃ and the rotation speed of 250r/min, wherein the concentration of nicotinamide adenine dinucleotide phosphate in the system reaches 95g/L, and the conversion rate reaches about 95%.

Example 9

Sodium trimetaphosphate 2800g, 672g NAD kinase, MgSO₄·7H₂O 0.7mol，CoCl₂0.42mol, PBS buffer solution (pH 7.0) is dissolved to 14L, substrate is added into the reaction system by a continuous flow adding mode, the addition amount of NAD is 1400g, the reaction is carried out for 3h at the temperature of 37 ℃ and the rotating speed of 250r/min, the concentration of nicotinamide adenine dinucleotide phosphate in the system reaches 98g/L, and the conversion rate reaches about 98.5 percent.

From the results of examples 5 to 9, it is understood that the inhibition effect on the enzymatic reaction is enhanced with the increase of the addition amount of substrate NAD, and when the addition amount in one time in the system reaches 200g, the inhibition effect is significant, resulting in the significant decrease of the conversion rate of the enzymatic reaction. The invention avoids the inhibition of the substrate on the reaction, keeps higher conversion rate and realizes industrialized production by changing the substrate adding mode and adopting the continuous feeding mode.

In addition, from the above-mentioned embodiments, Mg can be known²⁺、Co²⁺Has better activation effect on enzyme, when 0.05mol/L Mg is added²⁺And 0.03mol/L Co²⁺The activation effect is most remarkable. Meanwhile, the optimum conditions for the enzymatic reaction system are: NAD 10%, sodium trimetaphosphate 20%, enzyme base ratio 30:1, 0.05mol/L Mg²⁺，0.03mol/L Co²⁺The conversion rate of nicotinamide adenine dinucleotide phosphate can reach about 98.5% at the highest by fixing and dissolving in PBS buffer solution (pH 7.0), and reacting for 3h by continuously feeding substrate at the temperature of 37 ℃ and the rotating speed of 250 r/min.

According to the invention, through the research on an NAD kinase enzymatic mechanism, an enzyme activator with a proper concentration is screened and added, the activity of the enzyme is activated, and meanwhile, the enzyme plays a role of a bridge when the enzyme is combined with a substrate, on the basis, after the enzymatic reaction temperature, the substrate concentration, the enzyme substrate ratio, the substrate adding mode and the like are optimized, the concentration of nicotinamide adenine dinucleotide phosphate reaches 98g/L, the conversion rate reaches about 98.5 percent, and the nicotinamide adenine dinucleotide phosphate is the highest level reported in the current literature. An enzymatic reaction channel is constructed under the action of an enzyme activator, the enzymatic reaction time is shortened to 3h, meanwhile, the substrate adding mode is changed into continuous flow addition, the inhibition effect of the substrate on the reaction is avoided, and the high-conversion-rate production after the system is amplified is realized.

In addition, metaphosphate is used as a phosphate group donor instead of ATP, so that the cost of raw materials is greatly reduced, the operation is simple, the reaction is thorough, and the industrial and large-scale production is easy to realize.

In the description of the present invention, numerous specific details are set forth. It is understood, however, that embodiments of the invention may be practiced without these specific details. In some instances, well-known methods, structures and techniques have not been shown in detail in order not to obscure an understanding of this description.

The above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.