Nicotinamide enzyme mutant and method for producing nicotinic acid by using nicotinamide enzyme mutant

Abstract

The invention belongs to the technical field of enzyme engineering and gene engineering, and particularly relates to a nicotinamide enzyme mutant and application of the nicotinamide enzyme mutant in a method for producing nicotinic acid based on a nitrile hydratase nicotinamide enzyme cascade catalytic system. A rate-limiting enzyme of a reaction channel is firstly determined to be nicotinamide enzyme, a saturated mutant library is created through directed evolution, a series of nicotinamide enzyme mutants with high catalytic efficiency are obtained, and the A79S enzyme activity is improved most obviously and reaches 759.2 U / mg and is improved by 4.74 times compared with a wild type. Escherichia coli resting cells respectively containing nitrile hydratase and nicotinamide mutant genes are connected in series by utilizing a cascade reactor model, the optimal conditions of the two strains are controlled, finally efficient continuous synthesis of NA is realized, and the yield of nicotinic acid reaches 298.4 g / L. The nitrile hydratase and the nicotinamide enzyme can play the highest catalytic efficiency, the two catalysis reactions are complete, and the substrate inhibition effect is avoided at the same time.

Description

Technical Field

[0001] The invention relates to a nicotinamide enzyme mutant and an application of a nitrile hydratase-nicotinamide enzyme cascade system using 3-cyanopyridine as a substrate, and belongs to the technical field of enzyme engineering and genetic engineering. Background Art

[0002] Nicotinic acid, also known as vitamin B3, is an organic compound, a white crystalline powder, and one of the 13 vitamins necessary for the human body. Nicotinic acid is mainly used as a feed additive to improve the utilization rate of feed protein, increase the milk production of dairy cows, and increase the output and quality of fish, chicken, duck, cattle, sheep and other livestock meat. Nicotinic acid is also a widely used pharmaceutical intermediate. It can be used as a raw material to synthesize a variety of medicines, such as nikethamide and inositol nicotinate. In addition, niacin also plays an irreplaceable role in the fields of luminescent materials, dyes, and electroplating industries.

[0003] At present, the synthesis of NA is mainly through chemical synthesis, which has the disadvantages of low yield and high energy cost. The green biological method for synthesizing NA has attracted more and more attention and is expected to replace the traditional chemical synthesis route involving harsh conditions. Nitriles are widely distributed in the environment. In nature, they are produced by plants in various forms, such as cyanoglycosides and cyanolipids. They are also important intermediates for amines, carboxylic acids and heterocyclic compounds. They can be hydrolyzed into the corresponding acids and ammonia by nitrase through a single or continuous double enzyme pathway. 3-Cyanopyridine, also known as nicotinonitrile, can be converted into nicotinic acid by nitrilase (EC 3.5.5.1). Stenotrophomonas maltophilia AC21 used nitrilase to catalyze 116.2g / L NA in 10h by a batch feeding method. Gordonia terrae MN12 also used nitrilase to catalyze 221g / L NA in 270min by a batch feeding method with a catalytic efficiency of 24.56g / h / g dcw. However, nitrilase has problems such as relatively low catalytic activity, poor operational stability and limited substrate tolerance, which restricts the development of large-scale production of niacin.

[0004] 3-Cyanopyridine can also be converted to nicotinic acid by nitrile hydratase (EC 4.2.1.84) and nicotinamidase (EC 3.5.1.4). The former catalyzes the hydration of 3-cyanopyridine to nicotinamide, which is then converted to nicotinic acid by the latter. This sequential double enzyme hydrolysis ensures absolute selectivity. Microbacterium imperial CBS 498-74, using ultrafiltration membrane bioreactor (CSMR), achieved a 100% conversion rate of 3-cyanopyridine and obtained 23.73g / L NA, but some nicotinamide was not completely converted, which means that there is a problem of mismatch in the catalytic efficiency of the two enzymes. Summary of the invention

[0005] The invention aims to construct a chassis cell for efficiently synthesizing nicotinic acid by cascade reaction.

[0006] To achieve the above object, the present invention is implemented by the following scheme:

[0007] The amino acid sequence of the wild-type nicotinamidase strain is shown in SEQ ID NO.2. The amidase mutant was screened out from the wild-type strain by saturation mutagenesis and high-throughput screening. The amino acid sequence of the nicotinamidase mutant A79S is shown in SEQ ID NO.4. The wild nicotinamidase gene is from Mycobacterium tuberculosis, as described in the reference "Characterization of Mycobacterium tuberculosis nicotinamidase / pyrazinamidase. The FEBS Journal 2008, 275(4), 753-762".

[0008] The present invention provides a method for improving the activity of nicotinamide enzyme, which comprises mutating the alanine at position 79 of the nicotinamide enzyme of the amino acid sequence as shown in SEQ ID NO.2 into serine.

[0009] The wild nicotinamidase gene is shown in SEQ ID NO.1:

[0010] atgcgggcgttgatcatcgtcgacgtgcagaacgacttctgcgagggtggctcgctggcggtaaccggtggcgccgcgctggcccgcgccatcagcgactacctggccgaagcggcggactaccatcacgtcgtggcaaccaaggacttccacatcgacccgggtgaccacttctccggcacaccggactattcctcgtcgtggccaccgcattgcgtcagcggtactcccggcgcggacttccatcccagtctggacacgtcggcaatcgaggcggtgttctacaagggtgcctacaccggagcgtacagcggcttcgaaggagtcgacgagaacggcacgccactgctgaattggctgcggcaacgcggcgtcgatgaggtcgatgtggtcggtattgccaccgatcattgtgtgcgccagacggccgaggacgcggtacgcaatggcttggccaccagggtgctggtggacctgacagcgggtgtgtcggccgataccaccgtcgccgcgctggaggagatgcgcaccgccagcgtcgagttggtttgcagctcctga

[0011] The amino acid sequence of the wild-type nicotinamidase strain is shown in SEQ ID NO.2:

[0012] MRALIIVDVQNDFCEGGSLAVTGGAALARAISDYLAEAADYHHVVATKDFHIDPGDHFSGTPDYSSSWPPHCVSGTPGADFHPSLDTSAIEAVFYKGAYTGAYSGFEGVDENGTPLLNWLRQRGVDEVDVVGIATDHCVRQTAEDAVRNGLATRVLVDLTAGVSADTTVAALEEMRTASVELVCSS

[0013] The sequence of the coding gene of the amidase mutant is shown in SEQ ID NO.3:

[0014] atgcgggcgttgatcatcgtcgacgtgcagaacgacttctgcgagggtggctcgctggcggtaaccggtggcgccgcgctggcccgcgccatcagcgactacctggccgaagcggcggactaccatcacgtcgtggcaaccaaggacttccacatcgacccgggtgaccacttctccggcacaccggactattcctcgtcgtggccaccgcattgcgtcagcggtactcccggcttcgacttccatcccagtctggacacgtcggcaatcgaggcggtgttctacaagggtgcctacaccggagcgtacagcggcttcgaaggagtcgacgagaacggcacgccactgctgaattggctgcggcaacgcggcgtcgatgaggtcgatgtggtcggtattgccaccgatcattgtgtgcgccagacggccgaggacgcggtacgcaatggcttggccaccagggtgctggtggacctgacagcgggtgtgtcggccgataccaccgtcgccgcgctggaggagatgcgcaccgccagcgtcgagttggtttgcagctcctga

[0015] The amino acid sequence of the amidase mutant is shown in SEQ ID NO.4:

[0016] MRALIIVDVQNDFCEGGSLAVTGGAALARAISDYLAEAADYHHVVATKDFHIDPGDHFSGTPDYSSSWPPHCVSGTPGSDFHPSLDTSAIEAVFYKGAYTGAYSGFEGVDENGTPLLNWLRQRGVDEVDVVGIATDHCVRQTAEDAVRNGLATRVLVDLTAGVSADTTVAALEEMRTASVELVCSS。

[0017] Preferably, the optimal pH of the amidase mutant is 7.5. The optimal temperature of the amidase is 40 °C.

[0018] Further, the gene encoding nitrile hydratase and the gene encoding nicotinamide enzyme were connected to the pET-28a(+) plasmid respectively, and transformed into E. coli BL21(DE3). Two transformants were selected and inoculated into LB liquid medium with Kan resistance, cultured at 37°C for 12 hours, and transferred to 2×YT medium and TB medium with Kan resistance respectively, wherein the gene encoding nitrile hydratase was inoculated into 2×YT medium, and the gene encoding nicotinamide enzyme was inoculated into TB medium, and the inoculation amount was 5%. When the bacteria grew to OD 600 When the pH value was 0.6-0.8, IPTG was added for induction, and the culture temperature was lowered to 24°C for 24 hours. The supernatant was collected and purified to obtain nitrile hydratase and nicotinamide adenine enzyme mutants.

[0019] The nucleotide sequence of the nitrile hydratase is shown in SEQ ID NO.5, and the nitrile hydratase gene is from Caldalkalibacillus thermarum TA2.TA1, as recorded in the reference “Substrate access tunnel engineering for improving the catalytic activity of a thermophilic nitrilehydratase toward pyridine and pyrazine nitriles. Biochemical and Biophysical Research Communications 2021, 575, 8-13”.

[0020] The gene sequence of the nitrile hydratase mutant in the nitrile hydratase mutant strain is shown in SEQ ID NO.5:

[0021]

[0022] Another object of the present invention is to provide a method for efficiently producing niacin.

[0023] To achieve the above object, the present invention is implemented through the following scheme:

[0024] The optimal conditions for cell growth during the cultivation of recombinant strains containing mutant genes of nitrile hydratase and amidase were determined by shake flask catalysis. The optimal temperatures for cell growth during the cultivation of recombinant strains containing mutant genes of nitrile hydratase and amidase were 37°C and 40°C, respectively. The optimal pH for cell growth during the cultivation of recombinant strains containing mutant genes of nitrile hydratase and amidase were 7.0 and 7.4, respectively.

[0025] Cascade continuous catalysis is carried out in a 1.5L fermentation tank, including a first reactor and a second reactor forming a cascade system, a nitrile hydratase strain is arranged in the first reactor, and a nicotinamide enzyme mutant strain is arranged in the second reactor; the first reactor is connected to a feeding device, the first reactor and the second reactor are connected through a first pipeline, and the second reactor is connected to a nicotinic acid collector through a second pipeline; preferably, a peristaltic pump and a hollow fiber column are arranged on both the first pipeline and the second pipeline.

[0026] The optimum temperature and pH for nitrile hydratase / nicotinamidase catalysis are adjusted so that the enzymes operate with relatively unhindered kinetics.

[0027] Beneficial effects: The present invention mutates alanine in the nicotinamide enzyme molecule into serine by site-directed mutagenesis, changes the hydrophobicity and substrate interaction inside the molecule, and reshapes the active pocket to significantly improve the enzyme activity of the strain expressing amidase, reaching 759.2U / mg, which is 4.74 times higher than the wild type. By cascading continuous catalysis of the nitrile hydratase mutant strain and the amidase mutant strain, the optimal conditions of the two strains were controlled, and finally the efficient continuous synthesis of NA was achieved, and the nicotinic acid yield reached 298.4g / L. Both nitrile hydratase and nicotinamide enzyme can exert the highest catalytic efficiency, and both catalysis reactions are complete, while avoiding substrate inhibition and solving the problem of difficult product purification. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 :Distribution of nicotinamide enzyme active center and mutation points;

[0029] Figure 2 : Specific enzyme activity and fluorescence intensity of single-point mutants of nicotinamidase;

[0030] Figure 3 : Optimal temperatures of wild-type and mutant nicotinamidase;

[0031] Figure 4: Temperature stability of wild-type and mutant nicotinamidase;

[0032] Figure 5 : Optimal pH of wild-type and mutant nicotinamidase;

[0033] Figure 6 : pH stability of wild-type and mutant nicotinamidase;

[0034] Figure 7 :The hydrophobicity of wild-type and mutant nicotinamidase was measured by SDS method;

[0035] Figure 8 :The hydrophobicity of wild-type and mutant nicotinamidase was measured by bromophenol blue method;

[0036] Fig. 9 : The results of fed-batch catalysis of nitrile hydratase and nicotinamidase strains;

[0037] Fig.10 : Diagram of the cascade catalytic process of nitrile hydratase and nicotinamidase mutant strains;

[0038] Fig.11 : Cascade catalytic results of nitrile hydratase and nicotinamidase mutant strains. DETAILED DESCRIPTION

[0039] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0040] Example

[0041] LB medium: 10 g / L tryptone, 5 g / L yeast powder, 10 g / L NaCl, pH 7.4; solid medium plus 2% (w / v) agar on this basis; TB medium: 12 g / L tryptone, 24 g / L yeast powder, 9.4 g / L disodium hydrogen phosphate, 2.2 g / L dipotassium hydrogen phosphate, 4 ml / L glycerol, pH 7.4; 2×YT medium: 16 g / L tryptone, 10 g / L yeast powder, 5 g / L NaCl, pH7.4.

[0042] Phosphate buffer: 10 mM K2HPO4, 10 mM KH2PO4, pH 7.0.

[0043] Nicotinamide enzyme activity was determined by high performance liquid chromatography detection method. The catalytic activity of nicotinamide enzyme mutants was determined in 50mM potassium phosphate buffer (pH 7.5) with the enzyme activity of wild enzyme as 100%. The reaction volume was 1mL, inactivated with an equal volume of acetonitrile, and centrifuged at 12000rpm for 2min. The reaction system contained 20mM substrate 3-CN and 5μg purified recombinant protein. The sample was filtered through a 0.22μm filter and analyzed by HPLC system. The mobile phase was acetonitrile / water = 1:2 (v / v). The product was determined by HPLC at 230nm, and the enzyme activity was characterized by the product peak area.

[0044] 1. Construction of recombinant engineered bacteria

[0045] The nitrile hydratase gene was from C.thermarum TA2.TA1, and the nicotinamidase gene was from M.tuberculosis. They were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and cloned into the pET-28a(+) plasmid. Nitrile hydratase and nicotinamidase were heterologously expressed in Escherichia coli BL21(DE3).

[0046] 2. Screening of Nicotinamide Enzyme Mutant Strains

[0047] Nicotinamide enzyme forms a hydrophobic cavity when it works, and the hydrophobic effect drives the enzyme to maintain its natural formation, making the enzyme more stable. In this state, the enzyme catalytic activity is also higher, resulting in a higher V max Therefore, we speculated that the catalytic activity of nicotinamidase was related to the hydrophobicity of the protein, and analyzed the hydrophobicity of nicotinamidase using the ProtScale online tool.

[0048] In order to avoid affecting the normal function of the enzyme active center, Autodock was used to analyze the binding pocket and interaction between the nicotinamide enzyme mutation site and nicotinamide, and finally the distance from the active center was selected. Saturation mutations were performed on amino acids with high hydrophilicity in the range of 1:1. Figure 1 shown.

[0049] A random saturation mutant library of 10 sites was constructed, and the mutants in each library were identified using the nicotinic acid biosensor (NAsensor) screening platform. The nicotinamide enzyme mutant library was transformed into competent cells containing NAsensor, 30mmol / L nicotinamide was used as a substrate, 0.1mmol / L arabinose inducer was added, and it was spread on the LB plate. After culturing at 37°C for 16h, colonies with weaker fluorescence than the wild type were selected for pre-screening in a 96-well plate. The pre-screened strains were transferred to a 5mL test tube for rescreening, and the mutants with the most obvious inhibition of GFP expression were obtained and sequenced. The fluorescence intensities of mutants A79S and S67A were 13330.5 and 14814.8, respectively. The nicotinamide enzyme mutant gene fragment was connected to the vector pET28a(+), transformed into the BL21 expression host for induced expression and the specific enzyme activity was measured. The results are as follows Figure 2 As shown, a total of 16 mutants, including F13Y, F13G, G55C, G55H, G55P, G55S, S59A, S59Y, S66I, S67A, S67E, S67L, A79S, Y99H, and R140V, were obtained, among which the specific enzyme activities of mutants A79S and S67A reached 759.2 and 618.37 U / mg, respectively, which were 4.74 and 4.05 times higher than those of the wild type. The dominant mutant A79S was obtained, and its specific enzyme activity was 4.74 times higher than that of WT (wild type).

[0050] 3. Analysis of the enzymatic properties of WT and mutant nicotinamidase:

[0051] We investigated the optimal temperature and temperature stability of WT and mutants. Figure 3 As shown in the figure, the temperature detection range is 15-80℃. The catalytic activity of WT and mutants gradually increases with the increase of temperature. The optimum temperature of WT and S67A is 37℃, and the optimum temperature of A79S and S67A-A79S is 40℃. As the temperature continues to rise, their enzyme activities decrease rapidly. Incubate at the corresponding temperature for 20 minutes to explore the difference in temperature stability between WT and mutants. The results are shown in the figure. Figure 4 As shown, it was found that the stability of mutant A79S at 50-60°C was improved compared with the wild type. Secondly, we explored the optimal pH and pH stability of WT and mutants, such as Figure 5 As shown, the pH detection range is 3.0-11.0. The catalytic activity of WT and mutants gradually increases with the increase of pH. The optimal pH of WT and S67A is 7.0, and the optimal temperature of A79S and S67A-A79S is increased to 7.5 compared with the wild type. The pH stability test was determined after incubation at the corresponding pH for 20 minutes. The results are shown in Figure 6 The mutant A79S had a higher stability in the range of 7.5-9.0, indicating that the alkali resistance of A79S was improved.

[0052] We then measured the hydrophobicity of WT and mutant nicotinamide enzymes by SDS binding and bromophenol blue binding methods. SDS was added to the protein solution and adjusted to 0.07mM. The solution was allowed to stand for 30 minutes. After 24 hours of dialysis in a phosphate buffer solution with a pH of 6.0, 0.5ml of the dialysate was added to 10ml CHCl3. After mixing, 0.0024% methylene blue solution was added to the CHCl3 layer. After thorough mixing, the solution was centrifuged for 15 minutes. The SDS and methylene blue mixture at the bottom layer was taken and its absorbance was measured at a wavelength of 655nm. The results are shown in Table 1. Figure 7 The hydrophobicity of mutants S67A, A79S, and S67A-A79S were 2.3×10 5 , 2.2×10 5 , 2.1×10 5 Compared with WT, 1.87×10 5 They were 23%, 17.7%, and 12.3% higher, respectively, and then verified by the bromophenol blue binding method, such as Figure 8 The results were consistent with those of the SDS binding method, indicating that the enzymatic activity of nicotinamide enzyme was positively correlated with hydrophobicity within a certain range. At the same time, we determined the kinetic parameters of WT and mutants under optimal conditions, see Table 1. The Km values ​​of mutants S67A, A79S and S67A-A79S were 5.1, 3.4, and 3.8 mM, respectively, which were lower than 10.7 mM of WT, indicating that the affinity of the substrate to the mutants increased, and A79S had the highest affinity; the Kcat values ​​of mutants S67A, A79S and S67A-A79S were 10.9, 13.1, and 9.2 mM, respectively. -1 , which are 3.5, 4.2 and 3.0 times that of WT respectively. The catalytic efficiencies Kcat / Km of S67A, A79S and S67A-A79S are 7.1, 13 and 8.1 times that of WT respectively.

[0053] Table 1 Kinetic parameters of WT and its mutants

[0054]

[0055] 4. Batch production of niacin

[0056] The optimum temperature and pH of the mutants of nitrile hydratase and nicotinamide were determined by shake flask catalysis. The wild-type strain obtained 78.1 g / L NA and 68.3 g / L nicotinamide after terminating the reaction after 6 batches of feeding, while the mutant A79S strain obtained 143.3 g / L nicotinic acid after terminating the reaction after 6 batches of feeding, and no nicotinamide intermediates accumulated. The nicotinic acid production of the mutant A79S strain was 1.83 times that of the wild-type, indicating that the catalytic activity of the mutant A79S strain was much higher than that of the wild-type. Subsequently, the mixed strain was subjected to batch feeding catalysis in a 1.5L fermenter, and the results are shown in the figure. Fig. 9 As shown, the catalytic rate decreased when the seventh batch was fed, and a total of ten batches were fed. Finally, 224 g / L NA was obtained at a catalytic rate of 12.43 g / h / g dcw, and 13.3 g / L nicotinamide accumulated. In order to reduce the inhibition of 3-CN on nitrile hydratase and nicotinamide adenine, we used cascade fermentation to produce NA. Using cascade continuous catalytic reaction, the cascade catalytic process is as follows Fig.10 As shown, it is divided into two reactors, the first reactor 1 contains the nitrile hydratase strain, and the second reactor 2 contains the nicotinamide enzyme mutant strain (AMase mutant strain), which are connected by a hollow fiber column 3 to form a cascade system. The first peristaltic pump 6 set on the connecting pipe between the first reactor 1 and the feeding device 8 is adjusted to feed at a speed of 15mL / min to maintain a low concentration of 3-CN below 15g / L, and HPLC monitors the concentration change. At 40min, after 120g / L nicotinamide accumulates in the first reactor 1, the second peristaltic pump 5 set on the pipe between the first reactor 1 and the second reactor 2 is turned on to feed the reaction liquid in the first reactor 1 to the second reactor 2 containing the nicotinamide enzyme mutant at 40mL / min, and the nitrile hydratase bacteria in the reaction liquid in the first reactor 1 are refluxed to the first reactor 1 through the hollow fiber column 3 set on the pipe between the first reactor 1 and the second reactor 2. In order to prevent the liquid volume in the first reactor 1 from decreasing, the first reactor 1 is connected to the phosphate buffer feeding bottle to maintain the volume of the first reactor 1 at 1.1L. After 20 minutes, 73 g / L NA was accumulated in the second reactor 2. The third peristaltic pump 7 on the pipeline between the second reactor 2 and the nicotinic acid collector 4 was adjusted to run at a speed of 40 ml / min to continuously filter out the nicotinic acid clear liquid.

[0057] After nearly 1.5 hours of continuous synthesis of nicotinic acid, the reaction rate gradually decreased, and the reaction was finally stopped at 2.5 hours, and a total of about 298.4 g / L NA ( Fig.11 ), which is 33.2% higher than the yield of batch feeding. The rate of 3-CN to NA conversion per hour is about 85.26 g / L / h, which is obtained by dividing the total yield by the total time. The cell dry weight measured in this paper is about 3.4 mg dcw / mL. The catalytic efficiency of the cascade system is 25.46 g / h / g dcw, which is 104% higher than the catalytic efficiency of batch feeding.

[0058] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A nicotinamide enzyme mutant, wherein the nucleotide sequence of a wild nicotinamide enzyme gene is shown in SEQ ID NO.1, and the nicotinamide enzyme mutant is: the alanine at position 79 of the nicotinamide enzyme of the amino acid sequence shown in SEQ ID NO.2 is mutated to serine, and the amino acid sequence of the nicotinamide enzyme mutant is shown in SEQ ID NO.

4.

2. A gene encoding the mutant according to claim 1, wherein the nucleotide sequence of the gene is shown in SEQ ID NO.

3.

3. A recombinant plasmid carrying the gene according to claim 2.

4. A recombinant cell carrying the gene according to claim 2 or the recombinant plasmid according to claim 3.

5. The recombinant cell according to claim 4, characterized in that The pET-28a(+) plasmid was used as the connecting plasmid and Escherichia coli was used as the expression host.

6. Use of the mutant according to claim 1, or the gene according to claim 2, or the recombinant plasmid according to claim 3, or the recombinant cell according to claim 4 or 5 in the preparation of nicotinic acid.

7. A method for increasing nicotinic acid production, characterized in that: The alanine at position 79 of the nicotinamidase of the amino acid sequence shown in SEQ ID NO. 2 is mutated to serine.

8. A method for increasing nicotinic acid production, characterized in that: The nitrile hydratase strain and the nicotinamide enzyme mutant strain are subjected to cascade continuous catalysis; the gene sequence of the nitrile hydratase in the nitrile hydratase strain is shown as SEQ ID NO.5; the gene sequence of the nicotinamide enzyme mutant in the nicotinamide enzyme mutant strain is shown as SEQ ID NO.

3.

9. The method according to claim 1, characterized in that The nitrile hydratase strain is catalyzed in the first reactor, and the nicotinamide enzyme mutant strain is catalyzed in the second reactor; the two reactors are connected by a hollow fiber column and a peristaltic pump to form a cascade system; when nicotinamide accumulates to a certain amount in the first reactor, the reaction liquid in the first reactor is pumped into the second reactor through the hollow fiber column; after the reaction time is appropriate, the nicotinic acid clear liquid is continuously filtered out in the second reactor.

10. A niacin production system, characterized in that: The method comprises a first reactor and a second reactor, wherein the first reactor is connected to a feeding device, the first reactor and the second reactor are connected via a first pipeline, and the second reactor is connected to a nicotinic acid collector via a second pipeline; a peristaltic pump and a hollow fiber column are arranged on the first pipeline and the second pipeline; A nitrile hydratase strain is arranged in the first reactor, and a nicotinamide enzyme mutant strain is arranged in the second reactor; The gene sequence of nitrile hydratase in the nitrile hydratase strain is shown as SEQ ID NO.5; the gene sequence of nicotinamide enzyme mutant in the nicotinamide enzyme mutant strain is shown as SEQ ID NO.3.

Images