Method for preparing oxidized nicotinamide adenine dinucleotide phosphate through double-enzyme coupling

Abstract

The invention belongs to the field of biological catalysis, and particularly relates to a method for preparing oxidized nicotinamide adenine dinucleotide phosphate through double-enzyme coupling. According to the invention, through construction and optimization of a one-pot double-enzyme coupled catalytic system, a genetically engineered bacterium containing nicotinamide mononucleotide adenosine transferase NMNAT and a genetically engineered bacterium containing NAD kinase are used as a whole-cell catalyst, nicotinamide mononucleotide NMN is used as a starting substrate, adenosine triphosphate, polyphosphate and divalent metal ion salt are added, and the whole-cell enzyme is prepared through a one-pot method. The oxidized nicotinamide adenine dinucleotide phosphate NADP < + > is efficiently and catalytically synthesized by a one-pot method, and the whole-cell catalytic reaction liquid is separated and purified to obtain the NADP < + >. The double-enzyme catalysis method disclosed by the invention is short in reaction route, high in atom economy, capable of reducing the existing production cost, short in production period, easy for downstream separation and purification, and suitable for large-scale and large-scale production of NADP < + >.

Description

Technical Field

[0001] The invention belongs to the field of biocatalysis, and in particular relates to a method for preparing oxidized nicotinamide adenine dinucleotide phosphate by double enzyme coupling. Background Art

[0002] Nicotinamide adenine dinucleotide phosphate (NADP) + ) is a redox cofactor, mainly involved in cell anabolism, plays an important role in energy metabolism, and has the function of improving material transport and metabolism. Studies have shown that NADP + It can promote material metabolism, energy metabolism, resist cell aging and anti-oxidation. At the same time, since most of the endogenous coenzymes produced during the fermentation of engineered bacteria cannot meet the requirements of large-scale production, in most cases, additional coenzyme II needs to be added for redox reactions. NADP + It has a wide range of applications in enzyme catalysis, synthesis of chiral drugs and chiral intermediates. + The economic added value is high and the demand is large, so we develop NADP with high catalytic efficiency and low production cost. + Production technology has important economic value.

[0003] Currently synthesizing NADP + The main methods include chemical synthesis, biological fermentation, and biocatalysis. The chemical synthesis method has a long reaction route, involves the use of organic solvents, has poor selectivity, and is prone to produce by-products, causing environmental pollution problems. + The production process is relatively mature, but the atomic utilization rate is low and it is not conducive to the separation and purification of downstream products. The time-space yield is not high, the output is limited, and it is easy to increase the production cost. + It has the advantages of mild reaction conditions, high atom economy, and easy separation and purification, but it has defects such as raw material cost, time-space yield, and catalysis under high substrate concentration conditions, which limits the NADP + Large-scale and enlarged production. Summary of the invention

[0004] The present invention is to overcome the problem of synthesizing NADP in the prior art. + In order to overcome the defects of low conversion rate, long production cycle and complicated subsequent separation and purification steps, a method for preparing oxidized nicotinamide adenine dinucleotide phosphate by dual enzyme coupling is provided. The method uses nicotinamide mononucleotide (NMN) and adenosine triphosphate disodium salt (ATP·Na 2 ) as substrate, polyphosphate ((NaPO3 )n) is a phosphate group donor, a genetically engineered bacterium containing nicotinamide mononucleotide adenosyltransferase (NMNAT) and a genetically engineered bacterium containing NAD kinase are whole-cell catalysts, and NADP is synthesized in a one-pot method + .

[0005] To achieve the above-mentioned purpose, the present invention is implemented by the following technical solutions: A method for preparing oxidized nicotinamide adenine dinucleotide phosphate by dual enzyme coupling, comprising the following steps: Nicotinamide mononucleotide adenosine transferase (NMNAT) gene engineering bacteria and NAD kinase gene engineering bacteria were cultured and induced to express. The wet bacteria were obtained by centrifugation and preserved as whole-cell catalysts. Nicotinamide mononucleotide (NMN) and adenosine triphosphate (ATP·Na) disodium salt were used to synthesize the NMNAT gene engineering bacteria in the presence of divalent metal ions. 2 As a substrate, polyphosphate (NaPO 3 ) n As a phosphate group donor, a double enzyme reaction system is formed with a buffer as a reaction medium. After the reaction is complete, the reaction solution is separated and purified to obtain oxidized nicotinamide adenine dinucleotide phosphate NADP + .

[0006] The one-pot dual enzyme coupling catalytic system of the present invention comprises: wet bacteria obtained by fermentation of genetically engineered bacteria containing nicotinamide mononucleotide adenosine transferase NMNAT and genetically engineered bacteria containing NAD kinase as whole cell catalysts, nicotinamide mononucleotide NMN and adenosine triphosphate disodium salt ATP·Na 2 As a substrate, polyphosphate (NaPO 3 ) n As a phosphate group donor, a buffer solution is used as a reaction medium, and a divalent metal ion solution is added to form a reaction system. After the reaction is complete, the reaction solution is obtained and separated and purified to obtain the final product, namely, oxidized nicotinamide adenine dinucleotide phosphate NADP. + .

[0007] In order to improve the biocatalytic synthesis of NADP + The space-time yield is high, and the reaction cost is reduced. The present invention selects biocatalysis to synthesize NADP + The pathway is: through the dual enzyme coupling of nicotinamide mononucleotide adenylyltransferase (NMNAT) and NAD kinase, NMNAT catalyzes nicotinamide mononucleotide (NMN) to generate nicotinamide adenine dinucleotide (NAD + ) and pyrophosphate (PPi), while NAD kinase utilizes phosphate groups and NAD + NADP generation +NAD kinase can be divided into two categories according to the different phosphate group donors, one is ATP-dependent NAD kinase, and the other is ATP and Polyp (polyphosphate)-dependent NAD kinase, which can utilize both nucleoside triphosphates and inorganic polyphosphates. The dual enzyme coupling method established in the present invention is applied to NADP + The enzymatic synthesis has the advantages of low substrate cost and high catalytic efficiency. This dual enzyme coupling method has important research significance and practical application value.

[0008] On the other hand, the NMNAT single enzyme of the present invention catalyzes the synthesis of NAD + The reaction system is as follows: the wet bacteria obtained by fermentation of genetically engineered bacteria containing nicotinamide mononucleotide adenosine transferase NMNAT is used as the whole cell catalyst, and nicotinamide mononucleotide NMN and adenosine triphosphate disodium salt ATP·Na 2 As a substrate, a buffer solution with a pH of 6 to 9 is used as a reaction medium, a divalent metal ion solution is added to form a reaction system, and the reaction is carried out at 25 to 45°C and 100 to 800 rpm to obtain NAD + .

[0009] The NAD kinase of the present invention catalyzes the synthesis of NADP + The reaction system is as follows: the wet bacteria obtained by fermentation of genetically engineered bacteria containing NAD kinase is used as the whole cell catalyst, and nicotinamide adenine dinucleotide NAD + As a substrate, polyphosphate (NaPO 3 ) n As a phosphate group donor, a buffer solution with a pH of 6 to 9 is used as the reaction medium, a divalent metal ion solution is added to form a reaction system, and the reaction is carried out at 25 to 45°C and 100 to 800 rpm to obtain NADP + .

[0010] Preferably, the nicotinamide mononucleotide adenylyltransferase NMNAT genetically engineered bacteria is obtained by introducing a NMNAT mutant gene into Escherichia coli; the NMNAT mutant is derived from Methanothermobacterthermautotrophicus (thermoautotrophic methanobacterium), and has a double-point superposition mutation in which the 9th methionine in the amino acid sequence shown in SEQ ID NO.1 is mutated to glutamine and the 22nd arginine is mutated to glutamine, and the nucleotide sequence of the double-point superposition mutation is shown in SEQ ID NO.2.

[0011] Construction of Nicotinamide Mononucleotide Adenylyltransferase NMNAT Genetic Engineering Bacteria: The 9th methionine of the amino acid sequence shown in SEQ ID NO.1 was mutated to glutamine, and the 22nd arginine was mutated to glutamine by double-point superposition mutation, and the nicotinamide mononucleotide adenylyltransferase NMNAT mutant encoding gene was obtained, and the nucleotide sequence of the double-point superposition mutation was shown in SEQ ID NO. 2. The nicotinamide mononucleotide adenylyltransferase NMNAT mutant encoding gene shown in SEQ ID NO. 2 was inserted into the pET-28a vector and introduced into Escherichia coli to obtain the constructed gene.

[0012] Preferably, the NAD kinase genetically engineered bacteria is constructed by introducing a NAD kinase mutant gene into Escherichia coli; the NAD kinase mutant is derived from Saccharomyces cerevisiae (brewer's yeast), and is a double-point superposition mutation in which the arginine at position 252 of the amino acid sequence shown in SEQ ID NO.3 is mutated to asparagine, and the arginine at position 293 is mutated to alanine, and the nucleotide sequence of the double-point superposition mutation is shown in SEQ ID NO.4.

[0013] Construction of NAD kinase genetically engineered bacteria: The NAD kinase mutant encoding gene was obtained by double-point superposition mutation of the amino acid sequence shown in SEQ ID NO.3, arginine at position 252 to asparagine, and arginine at position 293 to alanine, from Saccharomyces cerevisiae (crysogenyces cerevisiae), and the nucleotide sequence of the double-point superposition mutation is shown in SEQ ID NO. 4. The NAD kinase mutant encoding gene shown in SEQ ID NO. 4 was inserted into the pET-28a vector and introduced into Escherichia coli to obtain the constructed gene.

[0014] Preferably, in a single-enzyme or dual-enzyme catalytic reaction system, the wet cells obtained by fermentation of genetically engineered bacteria containing nicotinamide mononucleotide adenylyltransferase NMNAT are prepared according to the following steps: The genetically engineered bacteria containing nicotinamide mononucleotide adenylyltransferase NMNAT were streaked and inoculated in an LB solid medium containing 50 mg / mL kanamycin, and cultured at 37°C for 12 hours; a single colony was picked and inoculated into an LB liquid medium containing 50 mg / mL kanamycin, and cultured at 180 rpm and 37°C for 7-8 hours, and then transferred to a TB liquid medium containing 50 mg / mL kanamycin at a volume concentration of 2%, and cultured in a shaking flask at 37°C and 180 rpm until the OD value reached 0.6-0.8, IPTG with a final concentration of 0.1 mM was added, and expression was induced at 28°C and 180 rpm for 12 hours, the bacterial solution was centrifuged at 8000 rpm for 10 minutes, the wet bacteria were collected, and stored at -20°C.

[0015] Preferably, in a single-enzyme or dual-enzyme catalytic reaction system, the wet cells obtained by fermentation of genetically engineered bacteria containing NAD kinase are prepared according to the following steps: The NAD kinase gene-engineered bacteria were streaked and inoculated in an LB solid culture medium containing 50 mg / mL streptomycin, and cultured at 37°C for 12 hours; a single colony was picked and inoculated into an LB liquid culture medium containing 50 mg / mL streptomycin, and cultured at 180 rpm and 37°C for 7-8 hours, and then transferred to a TB liquid culture medium containing 50 mg / mL streptomycin at a volume concentration of 2%, and cultured in a shaking flask at 37°C and 180 rpm until the OD value reached 0.6-0.8, IPTG with a final concentration of 0.1 mM was added, and expression was induced at 28°C and 180 rpm for 12 hours, the bacterial solution was centrifuged at 8000 rpm for 10 minutes, the wet bacteria were collected, and stored at -20°C.

[0016] Preferably, the reaction temperature of the NMNAT genetically engineered bacteria and the NAD kinase genetically engineered bacteria in the dual enzyme reaction system is 30-45° C., the reaction time is 1-12 h, and the rotation speed of the solution in the dual enzyme reaction system is 100-800 rpm.

[0017] As a further preference, the reaction temperature of the NMNAT genetically engineered bacteria and the NAD kinase genetically engineered bacteria in the dual enzyme reaction system is 37° C., the reaction time is 6 to 8 h, and the rotation speed of the solution in the dual enzyme reaction system is 650 rpm.

[0018] Preferably, the type of divalent metal ion in the dual enzyme reaction system is Zn 2+ Mg 2+ , Fe 2+ , Mn 2+ , Cu 2+ Any one or more combinations of .

[0019] As a further preferred embodiment, the type of divalent metal ion in the dual enzyme reaction system is Mg 2+ .

[0020] Preferably, the concentration of the divalent metal ions is 50 to 200 mM.

[0021] More preferably, the concentration of the divalent metal ions is 50 to 100 mM.

[0022] Preferably, the buffer is PB buffer or PBS buffer, and the pH of the buffer is 6-9.

[0023] As further preferred, the pH of the buffer solution is 7.5.

[0024] Preferably, in the dual enzyme reaction system, the concentration ratio of the wet bacteria containing nicotinamide mononucleotide adenylyltransferase NMNAT genetically engineered bacteria to the wet bacteria containing NAD kinase genetically engineered bacteria is 1:1.5-4.5; the added amount of the genetically engineered bacteria containing nicotinamide mononucleotide adenylyltransferase NMNAT and the genetically engineered bacteria containing NAD kinase is 30-60 g / L based on the total amount of wet bacteria.

[0025] As a further preference, the concentration ratio of the wet cells of the genetically engineered bacteria containing nicotinamide mononucleotide adenylyltransferase NMNAT to the wet cells of the genetically engineered bacteria containing NAD kinase is 1:2.

[0026] As a further preference, the added amount of the genetically engineered bacteria containing nicotinamide mononucleotide adenylyltransferase NMNAT and the genetically engineered bacteria containing NAD kinase is 30-45 g / L based on the total amount of wet bacteria.

[0027] Preferably, in the dual enzyme reaction system, the final concentration of the substrate nicotinamide mononucleotide NMN is 100-200 mM; the final concentration of nicotinamide mononucleotide NMN is 100-200 mM and the final concentration of adenosine triphosphate disodium salt ATP·Na 2 The final concentration ratio of nicotinamide mononucleotide NMN and polyphosphate (NaPO 3 ) n The final concentration ratio is 1:0.75~1.5.

[0028] As a further preferred embodiment, in the dual enzyme reaction system, the final concentration of the substrate nicotinamide mononucleotide NMN is 100-150 mM; the final concentration of nicotinamide mononucleotide NMN is 100-150 mM and the final concentration of adenosine triphosphate disodium salt ATP·Na 2 The final concentration ratio of nicotinamide mononucleotide NMN and polyphosphate (NaPO 3 ) n The final concentration ratio is 1:1.25.

[0029] Preferably, in the dual enzyme reaction system, the substrate is added in a manner of all at once or in two steps.

[0030] As a further preference, in the dual enzyme reaction system, the substrate is added all at once.

[0031] The NMN solution with equimolar concentration and the NMN-catalyzed NAD + The reaction liquid is a substrate solution, and the catalytic reaction is carried out under the same catalytic conditions. The space-time yield and substrate conversion rate of the one-time all-feeding method of the substrate are better than those of the two-step batch feeding method.

[0032] Preferably, the method for separating and purifying the reaction solution is: The reaction solution is treated with pyrophosphatase and then adjusted to pH 3-5. The bacteria are removed after centrifugation and the supernatant is taken. The clarified reaction solution is filtered and desalted to obtain a concentrated reaction solution. The concentrated reaction solution is freeze-dried to obtain NADP + Lyophilized powder, oxidized nicotinamide adenine dinucleotide phosphate NADP + .

[0033] As a further preference, the method for separating and purifying the reaction solution is: S1. Reaction solution pretreatment: Take the substrate that is completely converted and contains NADP + The reaction solution was treated with pyrophosphatase and 6 mol / L HCl solution was used to adjust the NADP + After the pH of the reaction solution was adjusted to 3-5, the bacteria were removed by centrifugation at 8000 rpm for 10 min, and then the NADP + The reaction solution is subjected to microfiltration and ultrafiltration to obtain a clarified reaction solution; S2, desalination treatment: The clarified reaction solution obtained in step S1 is subjected to nanofiltration using a 100D-500D nanofiltration membrane, or dialyzed in pure water using a 100D-500D semipermeable membrane for 12-48 hours under low temperature conditions to obtain desalted NADP + The reaction solution is concentrated to obtain a concentrated reaction solution; S3, freeze drying: The concentrated reaction solution obtained in step S2 was pre-frozen at -80°C for 12 hours, and placed in a vacuum freeze dryer for freeze drying for 24 hours to obtain NADP + Lyophilized powder, oxidized nicotinamide adenine dinucleotide phosphate NADP + .

[0034] As a further preferred method, the NADP-containing + The pH of the reaction solution is 3-7; the microfiltration membrane used in the microfiltration and ultrafiltration processes is a hollow fiber membrane with a pore size of 0.22-0.45 μm, and the ultrafiltration membrane is a hollow fiber membrane with a pore size of 3-10 kd.

[0035] As further preferred, the pore size of the nanofiltration membrane or semipermeable membrane used in step S2 is 300D.

[0036] More preferably, the concentration of the concentrated reaction solution obtained in step S2 is 150 to 180 g / L.

[0037] Therefore, the present invention has the following beneficial effects: (1) The present invention uses NMN as a starting substrate to prepare nicotinamide adenine dinucleotide phosphate NADP + , the reaction route is short, the catalytic efficiency is high, and the atom economy is high; in addition, the present invention uses polyphosphate instead of adenosine triphosphate as a phosphate group donor, which helps to reduce the cost of raw materials and is easy to realize industrial and large-scale production; (2) The present invention adopts a one-pot catalytic reaction, which has a thorough reaction and few by-products, and helps to improve the reaction efficiency and the NADP content in the reaction solution. + concentration, and no separation and purification of intermediate products is required. In addition, the present invention utilizes whole-cell catalysis, which is simple to operate, economical and efficient compared to microbial fermentation and pure enzyme catalysis, helps to improve the catalytic efficiency of the enzyme, reduces the enzyme purification process, and reduces production costs. Under the protection of the cell membrane, the intracellular enzyme has higher stability and longer half-life, and is easy to achieve high catalytic efficiency; (3) The present invention uses a membrane module separation method to purify NADP + , prepare NADP with purity above 90% from biocatalytic reaction liquid + The product has a yield of about 85%. The purification method is simple to operate, has little environmental pollution, reduces the generation of wastewater, reduces the use of organic solvents, and is suitable for large-scale and industrial separation and purification of NADP from whole-cell catalytic reaction liquid. + product. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 For the dual enzyme coupled catalytic synthesis of NADP + Method roadmap.

[0039] Figure 2 The figure is a bar graph showing the substrate conversion rate of NAD kinase using different phosphate group donors.

[0040] Figure 3 Effects of different temperatures on the catalytic activity of NMNAT and NAD kinase.

[0041] Figure 4 The effects of different metal ion activators on the catalytic activity of NMNAT and NAD kinase.

[0042] Figure 5 The effect of magnesium ion concentration on the catalytic enzyme activity of NMNAT and NAD kinase.

[0043] Figure 6 It is a single enzyme catalytic yield curve diagram of genetically engineered bacteria containing NNMAT and genetically engineered bacteria containing NAD kinase.

[0044] Figure 7 To investigate the effects of different substrate addition methods on dual-enzyme coupling catalytic reactions.

[0045] Figure 8 This is the effect of different bacterial concentrations on the dual-enzyme coupling catalytic reaction.

[0046] Fig. 9 The effect of different substrate concentrations on the dual enzyme coupled catalytic reaction.

[0047] Fig.10 It is a dual enzyme coupled catalytic synthesis of NADP + Time course graph.

[0048] Fig.11 For the separated and purified NADP + High performance liquid chromatography. DETAILED DESCRIPTION

[0049] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments of the specification. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments of the present invention, 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 invention.

[0050] The culture medium used in the embodiments of the present invention is composed as follows: LB liquid culture medium (g / L): peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, solvent is water, pH = 7.0, 121°C high pressure steam sterilization for 20 min.

[0051] LB solid medium is prepared by adding 20 g / L agar powder to LB liquid medium.

[0052] TB liquid culture medium (g / L): peptone 11.8 g / L, yeast extract 23.6 g / L, dipotassium hydrogen phosphate 9.4 g / L, potassium dihydrogen phosphate 2.2 g / L, glycerol 4 g / L, solvent is water, pH = 7.0, 121°C high pressure steam sterilization for 20 min.

[0053] 50 mg / mL kanamycin (Kan) stock solution: Weigh 0.5 g of kanamycin and place it on a clean bench. After ultraviolet sterilization for 15 to 20 minutes, dissolve it in 10 mL of sterile water. After sterilization, filter it with a 0.22 μm filter membrane and package it. Store it in a -20°C refrigerator for later use.

[0054] 50 mg / mL streptomycin (Str) stock solution: Weigh 0.5 g streptomycin and place it on a clean bench. After UV sterilization for 15 to 20 min, dissolve it in 10 mL sterile water. After sterilization, filter it with a 0.22 μm filter membrane and then divide it into packages. Store it in a -20°C refrigerator for later use.

[0055] 0.1 M isopropyl-β-D-thiogalactoside (IPTG) mother solution: Weigh 1.2 g IPTG and place it on a clean bench. After ultraviolet sterilization for about 15 minutes, dissolve it in 50 mL sterile water. After sterilization, filter it with a 0.22 μm filter membrane and package it. Store it in a -20°C refrigerator for later use.

[0056] In the embodiment of the present invention, NADP + The detection method is as follows: NADP + Detection method: Unitary C18 column (5μm, 100A, 4.6mm×250mm), mobile phase A: 50mM phosphate solution (disodium hydrogen phosphate: sodium dihydrogen phosphate = 5:4, pH adjusted to 6.8), mobile phase B: pure methanol, UV detector, wavelength 261nm, column temperature 30℃, flow rate 1mL / min, injection volume 10μL; retention time: NADP + About 9.7min, NAD + About 14.3 minutes.

[0057] In the embodiment of the present invention, NMNAT single enzyme catalyzes the synthesis of NAD + The reaction system is as follows: the wet bacteria obtained by fermentation of genetically engineered bacteria containing nicotinamide mononucleotide adenosine transferase NMNAT is used as the whole cell catalyst, and nicotinamide mononucleotide NMN and adenosine triphosphate disodium salt ATP·Na 2 As a substrate, a buffer solution with a pH of 6 to 9 is used as a reaction medium, a divalent metal ion solution is added to form a reaction system, and the reaction is carried out at 25 to 45°C and 100 to 800 rpm to obtain NAD + .

[0058] In the embodiments of the present invention, NAD kinase single enzyme catalyzes the synthesis of NADP + The reaction system is as follows: the wet bacteria obtained by fermentation of genetically engineered bacteria containing NAD kinase is used as the whole cell catalyst, and nicotinamide adenine dinucleotide NAD + As a substrate, polyphosphate (NaPO 3 ) nAs a phosphate group donor, a buffer solution with a pH of 6 to 9 is used as the reaction medium, a divalent metal ion solution is added to form a reaction system, and the reaction is carried out at 25 to 45°C and 100 to 800 rpm to obtain NADP + .

[0059] Example 1 This embodiment provides a method for preparing oxidized nicotinamide adenine dinucleotide phosphate by dual enzyme coupling.

[0060] A method for preparing oxidized nicotinamide adenine dinucleotide phosphate by dual enzyme coupling, comprising the following steps: Nicotinamide mononucleotide adenosine transferase (NMNAT) gene engineering bacteria and NAD kinase gene engineering bacteria were cultured and induced to express. The wet bacteria were obtained by centrifugation and preserved as whole-cell catalysts. Nicotinamide mononucleotide (NMN) and adenosine triphosphate (ATP·Na) disodium salt were used to synthesize the NMNAT gene engineering bacteria in the presence of divalent metal ions. 2 As a substrate, polyphosphate (NaPO 3 ) n As a phosphate group donor, a double enzyme reaction system is formed with a buffer as a reaction medium. After the reaction is complete, the reaction solution is separated and purified to obtain oxidized nicotinamide adenine dinucleotide phosphate NADP + . Synthesis of NADP by dual enzyme coupling + Method roadmap Figure 1 shown.

[0061] Among them, the type of divalent metal ion is Zn 2+ Mg 2+ , Fe 2+ , Mn 2+ , Cu 2+ Any one or more combinations of, most preferably Mg 2+ . The concentration of divalent metal ions is 50-200mM, most preferably 50-100mM. The buffer is PB buffer or PBS buffer, and the pH of the buffer is 6-9, most preferably 7.5. The concentration ratio of the wet bacteria containing the genetically engineered bacteria of nicotinamide mononucleotide adenosine transferase NMNAT and the wet bacteria containing the genetically engineered bacteria of NAD kinase is 1:1.5-4.5, most preferably 1:2. The amount of genetically engineered bacteria containing nicotinamide mononucleotide adenosine transferase NMNAT and genetically engineered bacteria containing NAD kinase added is 30-60g / L based on the total amount of wet bacteria, most preferably 30-45g / L. The final concentration of the substrate nicotinamide mononucleotide NMN is 100-200mM, most preferably 100-150mM. The final concentration of nicotinamide mononucleotide NMN is related to the disodium adenosine triphosphate ATP·Na 2The final concentration ratio of nicotinamide mononucleotide NMN to polyphosphate (NaPO 3 ) n The final concentration ratio is 1:0.75-1.5, and most preferably 1:1.25. The reaction temperature of the NMNAT genetic engineering bacteria and the NAD kinase genetic engineering bacteria in the dual enzyme reaction system is 30-45°C, and most preferably 37°C. The reaction time is 1-12h, and most preferably 6-8h. The rotation speed of the solution in the dual enzyme reaction system is 100-800rpm, and most preferably 650rpm.

[0062] Example 2: NAD kinase genetically engineered bacteria catalyze the synthesis of NADP using different phosphate group donors + Comparison of This example is to carry out single enzyme catalytic reaction on NAD kinase genetic engineering bacteria under the condition of equal substrate concentration using polyphosphate, adenosine triphosphate disodium salt, and a mixture of polyphosphate and adenosine triphosphate disodium salt. The wet cells of NAD kinase genetic engineering bacteria were prepared respectively by the following methods.

[0063] In a single-enzyme or dual-enzyme catalytic reaction system, wet cells obtained by fermentation of genetically engineered bacteria containing NAD kinase are prepared according to the following steps: The NAD kinase gene-engineered bacteria were streaked and inoculated in an LB solid culture medium containing 50 mg / mL streptomycin, and cultured at 37°C for 12 hours; a single colony was picked and inoculated into an LB liquid culture medium containing 50 mg / mL streptomycin, and cultured at 180 rpm and 37°C for 7-8 hours, and then transferred to a TB liquid culture medium containing 50 mg / mL streptomycin at a volume concentration of 2%, and cultured in a shaking flask at 37°C and 180 rpm until the OD value reached 0.6-0.8, IPTG with a final concentration of 0.1 mM was added, and expression was induced at 28°C and 180 rpm for 12 hours, the bacterial solution was centrifuged at 8000 rpm for 10 minutes, the wet bacteria were collected, and stored at -20°C.

[0064] In 10 mL phosphate buffer system, the substrate NAD + The final concentration of the phosphate group donors was 100 mM, the final concentration of polyphosphate was 80 mM, the final concentration of adenosine triphosphate disodium salt was 80 mM, the final concentration of the mixture of polyphosphate and adenosine triphosphate disodium salt was 80 mM, and the final concentration of Mg was 100 mM. 2+ The final concentration of the solution was 50 mM. The wet cells of the NAD kinase genetically engineered bacteria were resuspended in phosphate buffer to 20 g / L. Under the same other conditions of the enzymatic reaction, the reaction was continued for 8 h. The substrate conversion rates of the three phosphate group donors were 80.41%, 84.31%, and 86.75%, respectively. The substrate conversion rate of NAD kinase using different phosphate group donors is shown in the bar graph. Figure 2 shown.

[0065] from Figure 2 The analysis shows that the substrate conversion rate of the genetically engineered bacteria containing NAD kinase using a mixture of polyphosphate and disodium adenosine triphosphate as a phosphate group donor is higher than the substrate conversion rate of other polyphosphates or disodium adenosine triphosphate added alone as a phosphate group donor. Considering the raw material cost of the catalytic reaction, polyphosphate is selected as the phosphate group donor.

[0066] Example 3: Determination of the reaction temperature of the double enzyme coupling catalysis This example explores the effect of different temperatures on the catalytic reaction of NMNAT and NAD kinase. In the single enzyme catalytic system of NMNAT and NAD kinase, temperature gradients of 30°C, 34°C, 37°C, 40°C, 45°C, and 50°C were selected to investigate the effect of reaction temperature on the single enzyme catalytic reaction of NMNAT and NAD kinase, and the optimal reaction temperature was determined by relative activity. The effect of different temperatures on the catalytic enzyme activity of NMNAT and NAD kinase is shown in Figure 2. Figure 3 shown.

[0067] from Figure 3 From the analysis, we can see that the enzymatic reaction conversion rate of NMNAT is the highest when the reaction temperature is 45℃, and the enzymatic reaction conversion rate of NAD kinase is the highest when the reaction temperature is 37℃, thus determining that the optimal reaction temperature of the dual-enzyme coupled catalytic reaction system is 37℃.

[0068] Example 4: Effects of different metal ion activators and optimal metal ion concentrations on dual enzyme coupled catalytic reactions This example explores the effects of different types of metal ion activators and optimal metal ion concentrations on the catalytic reactions of NMNAT and NAD kinase. 2+ Mg 2+ , Fe 2+ , Mn 2+ and Cu 2+ The effect of divalent metal ions on the catalytic reaction was investigated by using divalent metal ion salts, and the optimal metal ion type and concentration were determined by relative activity. Figure 4 The effect of magnesium ion concentration on the catalytic activity of NMNAT and NAD kinase is shown in Figure 5 shown.

[0069] from Figures 4-5 The analysis shows that adding Mg 2+ It activates NMNAT and NAD kinase, among which Mg 2+ The activation effect is best when the added concentration is 0.05-0.1 mol / L.

[0070] Example 5: Determination of the substrate concentration range and bacterial concentration range of the dual enzyme coupling system This example explores the single enzyme catalytic reaction process of genetically engineered bacteria containing nicotinamide mononucleotide adenylyltransferase NMNAT and genetically engineered bacteria containing NAD kinase, so as to determine the substrate concentration range and bacterial concentration range of the dual enzyme coupling. The wet bacterial cells of NMNAT and NAD kinase were prepared by the following method.

[0071] In a single-enzyme or dual-enzyme catalytic reaction system, wet cells obtained by fermentation of genetically engineered bacteria containing nicotinamide mononucleotide adenylyltransferase NMNAT are prepared according to the following steps: The genetically engineered bacteria containing nicotinamide mononucleotide adenylyltransferase NMNAT were streaked and inoculated in an LB solid medium containing 50 mg / mL kanamycin, and cultured at 37°C for 12 hours; a single colony was picked and inoculated into an LB liquid medium containing 50 mg / mL kanamycin, and cultured at 180 rpm and 37°C for 7-8 hours, and then transferred to a TB liquid medium containing 50 mg / mL kanamycin at a volume concentration of 2%, and cultured in a shaking flask at 37°C and 180 rpm until the OD value reached 0.6-0.8, IPTG with a final concentration of 0.1 mM was added, and expression was induced at 28°C and 180 rpm for 12 hours, the bacterial solution was centrifuged at 8000 rpm for 10 minutes, the wet bacteria were collected, and stored at -20°C.

[0072] In a single-enzyme or dual-enzyme catalytic reaction system, wet cells obtained by fermentation of genetically engineered bacteria containing NAD kinase are prepared according to the following steps: The NAD kinase gene-engineered bacteria were streaked and inoculated in an LB solid culture medium containing 50 mg / mL streptomycin, and cultured at 37°C for 12 hours; a single colony was picked and inoculated into an LB liquid culture medium containing 50 mg / mL streptomycin, and cultured at 180 rpm and 37°C for 7-8 hours, and then transferred to a TB liquid culture medium containing 50 mg / mL streptomycin at a volume concentration of 2%, and cultured in a shaking flask at 37°C and 180 rpm until the OD value reached 0.6-0.8, IPTG with a final concentration of 0.1 mM was added, and expression was induced at 28°C and 180 rpm for 12 hours, the bacterial solution was centrifuged at 8000 rpm for 10 minutes, the wet bacteria were collected, and stored at -20°C.

[0073] In 10 mL phosphate buffer system, the final concentration of substrate NMN was 200 mM, the ratio of the final concentration of NMN to the final concentration of adenosine triphosphate disodium salt was 1:1.15, and the Mg 2+ The final concentration of the solution was 50 mM. The wet bacteria containing nicotinamide mononucleotide adenylyltransferase NMNAT were resuspended to 20 g / L with phosphate buffer. The temperature was 37° C., the rotation speed was 650 rpm, the reaction was carried out for 3 hours, and the substrate conversion rate was 97.15%.

[0074] In 10mL phosphate buffer system, substrate NAD + The final concentration was 200 mM, NAD + The final concentration of Mg was 1:1 with that of sodium polyphosphate. 2+ The final concentration of the solution was 50 mM, and the wet bacteria containing NAD kinase were resuspended in phosphate buffer to 20 g / L. The temperature was 37°C, the rotation speed was 650 rpm, and the reaction was carried out for 6 hours. The substrate conversion rate was 53.17%. The single enzyme catalytic yield curve of the genetically engineered bacteria containing NNMAT and the genetically engineered bacteria containing NAD kinase is shown in the figure. Figure 6 shown.

[0075] from Figure 6 The analysis shows that the engineered bacteria containing NMNAT can achieve 97% conversion of 200mM substrate under the catalysis of 20g / L bacterial concentration, and the engineered bacteria containing NAD kinase can achieve complete conversion of 100mM substrate under the catalysis of 20g / L bacterial concentration. According to the single enzyme catalytic reaction of the engineered bacteria containing NMNAT and the engineered bacteria containing NAD kinase on the substrate under the addition of 20g / L wet bacterial cells, the substrate concentration range of the dual enzyme coupling is determined to be 100mM to 200mM and the total wet bacterial concentration range is 30 to 60g / L.

[0076] Example 6: Effects of different substrate addition methods on dual enzyme coupled catalytic reactions In a 10 mL phosphate buffer system, the substrate was added all at once, the final concentration of the substrate NMN solution was 100 mM, and the substrate ATP·Na 2 The final concentration of the solution was 115 mM, the final concentration of the sodium polyphosphate solution was 100 mM, and the Mg 2+ The final concentration of the solution was 100 mM, the final concentrations of the genetically engineered bacteria containing nicotinamide mononucleotide adenylyltransferase NMNAT and the genetically engineered bacteria containing NAD kinase were 10 g / L and 20 g / L, respectively. The temperature was 37 °C, the rotation speed was 650 rpm, the reaction time was 6 h, the substrate conversion rate was 94.34%, and the space-time yield was 12.43 g*L -1 *h -1 .

[0077] In a 10 mL phosphate buffer system, the substrate was added in two steps in batches. First, NMN was used as a substrate to prepare a high concentration of NAD-containing + The specific reaction system is as follows: in 10 mL of phosphate buffer system, the final concentration of substrate NMN is 250 mM, the ratio of the final concentration of NMN to the final concentration of adenosine triphosphate disodium salt is 1:1.15, and the Mg 2+The final concentration of the solution was 100 mM, the final concentration of the genetically engineered bacteria containing nicotinamide mononucleotide adenylyltransferase NMNAT was 30 g / L, the temperature was 37 °C, the speed was 650 rpm, the reaction was carried out for 3 h, and the NAD + The reaction solution was centrifuged at 8000 rpm for 10 min to remove the bacteria and then stored at -20 °C for later use. In 10 mL of phosphate buffer system, the substrate NAD + The final concentration of the solution was 100 mM, the final concentration of the polyphosphate solution was 100 mM, and the Mg 2+ The final concentration of the solution was 100 mM, the final concentration of the NAD kinase-containing genetically engineered bacteria was 20 g / L, the temperature was 37 °C, the rotation speed was 650 rpm, the reaction time was 6 h, the substrate conversion rate was 84.24%, and the space-time yield was 8.32 g*L -1 *h -1 The effects of different substrate addition methods on the dual enzyme coupling catalytic reaction are as follows: Figure 7 shown.

[0078] from Figure 7 From the analysis, it can be seen that the one-time feeding method is superior to the two-step batch feeding method in terms of time-space yield and substrate conversion rate. The most suitable substrate addition method is to feed all at once.

[0079] Example 7: Effects of different bacterial concentrations on dual enzyme coupled catalytic reactions When other conditions of the enzymatic reaction are the same, the final concentration of the substrate NMN solution is 200 mM, and the concentration of ATP·Na 2 The final concentration of the solution was 250 mM, the final concentration of the polyphosphate solution was 250 mM, and the Mg 2+ The final concentration of the solution was 100mM, the temperature was 37℃, the speed was 650rpm, and the reaction time was 6h. The added concentrations of genetically engineered bacteria containing nicotinamide mononucleotide adenylyltransferase NMNAT and genetically engineered bacteria containing NAD kinase were 10g / L 20g / L, 15g / L 30g / L, 20g / L 40g / L, and the substrate conversion rates were 55.96%, 62.67%, and 59.35% respectively. The effects of different bacterial concentrations on the dual enzyme coupling catalytic reaction are as follows Figure 8 shown.

[0080] from Figure 8 The analysis shows that when the concentrations of the two genetically engineered bacteria were 15 g / L and 30 g / L, the catalytic reaction synthesized NADP. + The substrate conversion rate is the highest.

[0081] Example 8: Effect of different substrate concentrations on dual enzyme coupled catalytic reaction When other conditions of the enzymatic reaction are the same, the final concentration of the substrate NMN solution is: ATP·Na 2The final concentration of the solution was 1:1.25, the final concentration of the substrate NMN solution: the final concentration of the polyphosphate solution was 1:1.25, and the final concentration of Mg 2+ The final concentration of the solution was 100mM, and the concentrations of the genetically engineered bacteria containing nicotinamide mononucleotide adenylyltransferase NMNAT and the genetically engineered bacteria containing NAD kinase were 10g / L and 20g / L, respectively. The temperature was 37°C, the speed was 650rpm, and the reaction time was 6h. The final concentrations of the substrate NMN solution were 100mM, 150mM, and 200mM, respectively, and the product concentrations were 97.34mM, 123.69mM, and 111.91mM, respectively. The effects of different substrate concentrations on the dual enzyme coupling catalytic reaction are as follows Fig. 9 shown.

[0082] from Fig. 9 From the analysis, it can be seen that when the final concentration of the substrate NMN solution is 150mM, the yield of the dual-enzyme coupling catalytic reaction is the highest.

[0083] Example 9: Synthesis of NADP by dual enzyme coupling system + The reaction process This example is a dual enzyme coupled catalytic synthesis of NADP in a 10 mL reaction system when the substrate concentration is 100 mM. + The final concentration of the substrate NMN solution was 100 mM, ATP·Na 2 The final concentration of the solution was 115 mM, the final concentration of the polyphosphate solution was 100 mM, and the Mg 2+ The final concentration of the solution was 100mM, the final concentrations of the genetically engineered bacteria containing nicotinamide mononucleotide adenylyltransferase NMNAT and the genetically engineered bacteria containing NAD kinase were 10g / L and 20g / L, respectively. The temperature was 37°C, the speed was 650rpm, and the samples were taken for testing at 2, 4, 6, 8, and 10 hours. + The time course curve is as follows Fig.10 shown.

[0084] from Fig.10 From the analysis, we can see that as the reaction proceeds, the substrate is gradually consumed and the conversion rate increases accordingly. When the catalytic reaction time is 10h, the conversion rate of the substrate reaches 96.15%.

[0085] Example 10: NADP + Separation and purification The reaction solution was prepared according to the method in Example 1. This example is to prepare the product NADP by freeze drying after desalting the reaction solution. + The substrate was completely transformed and centrifuged to remove the NADP-containing + 500mL of reaction solution, in which NADP was detected +The content was 71.57%. After being treated with pyrophosphohydrolase, the pH was adjusted to 5.0 with 6 mol / L HCl solution and centrifuged at 8000 rpm for 10 min to obtain the pretreated NADP + Reaction solution; pre-treated NADP + The reaction liquid is passed through a microfiltration membrane (the microfiltration membrane is a hollow fiber membrane with a pore size of 0.22 to 0.45 μm) and an ultrafiltration membrane (the ultrafiltration membrane is a hollow fiber membrane with a pore size of 3 to 10 kd) to obtain a clarified reaction liquid from which macromolecular impurities are removed; the clarified reaction liquid is subjected to nanofiltration desalination using a 100D to 500D nanofiltration membrane (it can also be dialyzed in pure water under low temperature conditions for 12 to 48 hours using a 100D to 500D semipermeable membrane; the pore size of the nanofiltration membrane or the semipermeable membrane is most preferably 300D) to obtain desalted NADP + The reaction solution was concentrated to obtain a concentrated reaction solution; the concentrated reaction solution after desalination was pre-frozen at -80°C for 12 hours, placed in a vacuum freeze dryer for freeze drying for 24 hours, and NADP was obtained. + Lyophilized powder, oxidized nicotinamide adenine dinucleotide phosphate NADP + Product. NADP after separation and purification + High performance liquid chromatography Fig.11 As shown. NADP was detected by high performance liquid chromatography + The purity of the product was 92.39%.

[0086] The above description is only a detailed description of the preferred embodiments and principles of the present invention. For ordinary technicians in this field, according to the ideas provided by the present invention, there will be changes in the specific implementation methods, and these changes should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing oxidized nicotinamide adenine dinucleotide phosphate by dual enzyme coupling, characterized in that: The following steps are involved: Nicotinamide mononucleotide adenosine transferase (NMNAT) gene engineering bacteria and NAD kinase gene engineering bacteria were cultured and induced to express. Wet cells were obtained by centrifugation and preserved as whole-cell catalysts. Nicotinamide mononucleotide (NMN) and adenosine triphosphate disodium salt (ATP·Na2) were used as substrates in the presence of divalent metal ions, and polyphosphate (NaPO3) was used as catalyst. n As a phosphate group donor, a double enzyme reaction system is formed with a buffer as a reaction medium. After the reaction is complete, the reaction solution is separated and purified to obtain oxidized nicotinamide adenine dinucleotide phosphate NADP + .

2. The method for preparing oxidized nicotinamide adenine dinucleotide phosphate by dual enzyme coupling according to claim 1, characterized in that: The nicotinamide mononucleotide adenylyltransferase NMNAT genetic engineering bacteria is obtained by introducing the NMNAT mutant gene into Escherichia coli; the NMNAT mutant is derived from Methanothermobacterthermautotrophicus (thermoautotrophic methanobacterium), and is a double-point superposition mutation in which the 9th methionine of the amino acid sequence shown in SEQ ID NO.1 is mutated to glutamine and the 22nd arginine is mutated to glutamine, and the nucleotide sequence of the double-point superposition mutation is shown in SEQ ID NO.

2.

3. The method for preparing oxidized nicotinamide adenine dinucleotide phosphate by dual enzyme coupling according to claim 1, characterized in that: The NAD kinase genetic engineering bacteria is obtained by introducing the NAD kinase mutant gene into Escherichia coli; the NAD kinase mutant is derived from Saccharomyces cerevisiae (brewing yeast), and is a double-point superposition mutation in which the arginine at position 252 of the amino acid sequence shown in SEQ ID NO.3 is mutated to asparagine, and the arginine at position 293 is mutated to alanine, and the nucleotide sequence of the double-point superposition mutation is shown in SEQ ID NO.

4.

4. The method for preparing oxidized nicotinamide adenine dinucleotide phosphate by dual enzyme coupling according to claim 1, characterized in that: The reaction temperature of the NMNAT genetic engineering bacteria and the NAD kinase genetic engineering bacteria in the double enzyme reaction system is 30-45° C., the reaction time is 1-12 hours, and the rotation speed of the solution in the double enzyme reaction system is 100-800 rpm.

5. The method for preparing oxidized nicotinamide adenine dinucleotide phosphate by dual enzyme coupling according to claim 1, characterized in that: The type of divalent metal ion in the dual enzyme reaction system is Zn 2+ Mg 2+ , Fe 2+ , Mn 2+ , Cu 2+ Any one or more combinations of .

6. The method for preparing oxidized nicotinamide adenine dinucleotide phosphate by dual enzyme coupling according to claim 1 or 5, characterized in that: The concentration of divalent metal ions is 50-200 mM.

7. The method for preparing oxidized nicotinamide adenine dinucleotide phosphate by dual enzyme coupling according to claim 1, characterized in that: The buffer is a PB buffer or a PBS buffer, and the pH of the buffer is 6-9.

8. The method for preparing oxidized nicotinamide adenine dinucleotide phosphate by dual enzyme coupling according to claim 1, characterized in that: In the dual enzyme reaction system, the concentration ratio of the wet bacteria containing nicotinamide mononucleotide adenosyltransferase NMNAT gene engineering bacteria and the wet bacteria containing NAD kinase gene engineering bacteria is 1:1.5-4.5; the added amount of the nicotinamide mononucleotide adenosyltransferase NMNAT gene engineering bacteria and the NAD kinase gene engineering bacteria is 30-60g / L based on the total amount of wet bacteria.

9. The method for preparing oxidized nicotinamide adenine dinucleotide phosphate by dual enzyme coupling according to claim 1, characterized in that: In the dual enzyme reaction system, the final concentration of the substrate nicotinamide mononucleotide NMN is 100-200 mM; the ratio of the final concentration of nicotinamide mononucleotide NMN to the final concentration of adenosine triphosphate disodium salt ATP·Na2 is 1:1-2; the final concentration of nicotinamide mononucleotide NMN is 1:1-2 to the final concentration of polyphosphate (NaPO3) n The final concentration ratio is 1:0.75~1.

5.

10. The method for preparing oxidized nicotinamide adenine dinucleotide phosphate by dual enzyme coupling according to claim 1, characterized in that: The method for separating and purifying the reaction solution is: The reaction solution is treated with pyrophosphatase and then adjusted to pH 3-5. The bacteria are removed after centrifugation and the supernatant is taken. The clarified reaction solution is filtered and desalted to obtain a concentrated reaction solution. The concentrated reaction solution is freeze-dried to obtain NADP + Lyophilized powder, oxidized nicotinamide adenine dinucleotide phosphate NADP + .

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