Nicotinamide enzyme mutants and their methods in the production of nicotinic acid

Abstract

This invention belongs to the fields of enzyme engineering and genetic engineering, specifically relating to nicotinamide enzyme mutants and their application in a method for producing nicotinic acid based on a nitrile hydratase-nicotinamide enzyme cascade catalytic system. The invention first identifies nicotinamide enzyme as the rate-limiting enzyme in the reaction pathway. Through directed evolution, a saturated mutant library is created, yielding a series of nicotinamide enzyme mutants with high catalytic efficiency. Among them, the A79S mutant shows the most significant increase in enzyme activity, reaching 759.2 U / mg, a 4.74-fold increase compared to the wild type. Subsequently, using a cascade reactor model, resting *E. coli* cells containing nitrile hydratase and nicotinamide enzyme mutant genes are connected in series. By controlling the optimal conditions for both strains, highly efficient continuous synthesis of NA is achieved, with a nicotinic acid yield of 298.4 g / L. Both nitrile hydratase and nicotinamide enzyme exhibit maximum catalytic efficiency, and both catalytic reactions are complete, while substrate inhibition is avoided.

Description

Technical Field

[0001] This invention relates to the application of nicotinamide mutants and nitrile hydratase-nicotinamide cascade systems based on 3-cyanopyridine, belonging to the fields of enzyme engineering and genetic engineering technology. Background Technology

[0002] Niacin, also known as vitamin B3, is an organic compound, a white crystalline powder, and one of the 13 essential vitamins for the human body. Niacin is mainly used as a feed additive to improve the utilization rate of feed protein, increasing milk production in dairy cows and the yield and quality of meat from poultry and livestock such as fish, chicken, duck, beef, and mutton. Niacin is also a widely used pharmaceutical intermediate; it can be used to synthesize various pharmaceuticals, such as nikethamide and nicotinic acid inositol ester. Furthermore, nicotinic acid plays an irreplaceable role in the fields of luminescent materials, dyes, and electroplating.

[0003] Currently, nitriles (NA) are mainly synthesized through chemical synthesis, which suffers from drawbacks such as low yield and high energy consumption. Green biological methods for NA synthesis are gaining increasing attention and hold promise for replacing traditional chemical synthesis routes involving harsh conditions. Nitriles are widely distributed in the environment. In nature, they are produced by plants in various forms, such as cyanoglycosides and cyanoesters. They are also important intermediates for amines, carboxylic acids, and heterocyclic compounds. They can be hydrolyzed by nitrases through single or continuous dual-enzyme pathways to the corresponding acids and ammonia. 3-Cyanopyridine, also known as nicotinic nitrile, can be converted to nicotinic acid by nitrile hydrolase (EC 3.5.5.1). Stenotrophomonas maltophilia AC21 used nitrile hydrolase in a fed-batch manner to produce 116.2 g / L NA in 10 h. Gordonia terrae MN12 also used nitrile hydrolase in a fed-batch manner to produce 221 g / L NA in 270 min with a catalytic efficiency of 24.56 g / h / g dcw. However, nitrile hydrolases have problems such as relatively low catalytic activity, poor operational stability, and limited substrate tolerance, which restricts the development of large-scale nicotinic acid production.

[0004] 3-Cyanopyridine can also be converted to nicotinic acid by nitrile hydratase (EC 4.2.1.84) and nicotinamide enzyme (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 two-enzyme hydrolysis ensures absolute selectivity. Using a Microbacterium imperial CBS 498-74 ultrafiltration membrane bioreactor (CSMR), 100% conversion of 3-cyanopyridine was achieved, yielding 23.73 g / L NA. However, some nicotinamide was not completely converted, indicating a mismatch in the catalytic efficiencies of the two enzymes. Summary of the Invention

[0005] The purpose of this invention is to construct a chassis cell for the efficient synthesis of nicotinic acid via a cascade reaction.

[0006] To achieve the above objectives, the present invention is implemented through the following solution:

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

[0008] This invention provides a method for improving nicotinamide enzyme activity by mutating alanine at position 79 of the nicotinamide enzyme, as shown in SEQ ID NO.2, to serine.

[0009] The wild-type nicotinamide enzyme 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 optimum pH of the amidase mutant is 7.5. The optimum temperature of the amidase is 40°C.

[0018] Furthermore, the genes encoding nitrile hydratase and nicotinamide enzyme were ligated into the pET-28a(+) plasmid and transformed into *E. coli* BL21(DE3). Two transformants were selected and inoculated into LB liquid medium containing Kan resistance, cultured at 37°C for 12 h, and then transferred to Kan-resistant 2×YT and TB media, respectively. The transformant containing the nitrile hydratase gene was inoculated into 2×YT medium, and the transformant containing the nicotinamide enzyme gene was inoculated into TB medium, with an inoculation volume of 5%. The cells were cultured until they reached OD... 600 When the pH was 0.6-0.8, IPTG was added for induction, and the culture temperature was lowered to 24℃ and cultured for 24 h. The supernatant was collected and purified to obtain nitrile hydratase and nicotinamide enzyme mutants.

[0019] The nucleotide sequence of the nitrile hydratase is shown in SEQ ID NO.5. The nitrile hydratase gene is derived from Caldalkalibacillus thermarum TA2.TA1, as described 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 strain is shown in SEQ ID NO. 5:

[0021]

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

[0023] To achieve the above objectives, the present invention is implemented through the following solution:

[0024] The optimal cell growth conditions for recombinant strains containing nitrile hydratase and amidase mutant genes were determined using shake-flask catalysis. The optimal temperatures for cell growth during the culture of recombinant strains containing nitrile hydratase and amidase mutant genes were 37℃ and 40℃, respectively. The optimal pH values ​​for cell growth during the culture of recombinant strains containing nitrile hydratase and amidase mutant genes were 7.0 and 7.4, respectively.

[0025] Cascaded continuous catalysis is carried out in a 1.5L fermenter, including a first reactor and a second reactor forming a cascade system. The first reactor is equipped with a nitrile hydratase strain, and the second reactor is equipped with a nicotinamide enzyme mutant strain. The first reactor is connected to a feeding device, and the first reactor and the second reactor are connected through a first pipe. The second reactor is connected to a nicotinic acid collector through a second pipe. Preferably, both the first pipe and the second pipe are equipped with peristaltic pumps and hollow fiber columns.

[0026] Adjusting the optimal temperature and pH for nitrile hydratase / nicotinamide enzyme catalysis allows the enzyme to operate with relatively unimpeded kinetics.

[0027] Beneficial Effects: This invention utilizes site-directed mutagenesis to mutate alanine into serine within the nicotinamide enzyme molecule, altering the internal hydrophobicity and substrate interactions. Furthermore, the remodeling of the active pocket significantly enhances the enzyme activity of the expressed amidase, reaching 759.2 U / mg, a 4.74-fold increase compared to the wild type. By cascading the nitrile hydratase mutant strain and the amidase mutant strain for continuous catalysis, and controlling the optimal conditions for both strains, highly efficient continuous synthesis of NA was achieved, with a nicotinic acid yield of 298.4 g / L. Both nitrile hydratase and nicotinamide enzyme exhibited maximum catalytic efficiency, resulting in complete reactions while avoiding substrate inhibition and resolving the difficulty of product purification. Attached Figure Description

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

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

[0030] Figure 3 Optimal temperatures for wild-type and mutant nicotinamide enzymes;

[0031] Figure 4Temperature stability of wild-type and mutant nicotinamide enzymes;

[0032] Figure 5 Optimal pH for wild-type and mutant nicotinamide enzymes;

[0033] Figure 6 pH stability of wild-type and mutant nicotinamide enzymes;

[0034] Figure 7 SDS method for determining the hydrophobicity of wild-type and mutant nicotinamide enzymes;

[0035] Figure 8 : Determination of hydrophobicity of wild-type and mutant nicotinamide enzymes using the bromophenol blue method;

[0036] Figure 9 Results of fed-batch catalysis of nitrile hydratase and nicotinamide enzyme strains;

[0037] Figure 10 : Cascade catalytic process diagram of nitrile hydratase and nicotinamide enzyme mutant strains;

[0038] Figure 11 Results of cascade catalysis by mutant strains of nitrile hydratase and nicotinamide. Detailed Implementation

[0039] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0040] Example

[0041] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.4; solid medium is prepared with 2% (w / v) agar added; TB medium: 12 g / L tryptone, 24 g / L yeast extract, 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 extract, 5 g / L NaCl, pH 7.4.

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

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

[0044] 1. Construction of recombinant engineered bacteria

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

[0046] 2. Screening of Nicotinamide Mutant Strains

[0047] Nicotinamide enzymes form hydrophobic cavities during their function, and this hydrophobic effect drives the enzyme to maintain its native formation, making it more stable. In this state, the enzyme's catalytic activity is also higher, resulting in the production of higher V. max Therefore, we hypothesized that the catalytic activity of nicotinamide is related to protein hydrophobicity, and thus analyzed the hydrophobicity of nicotinamide using the ProtScale online tool.

[0048] To avoid affecting the normal function of the enzyme's active site, Autodock analysis was used to examine the binding pocket and interaction between the nicotinamide mutation site and nicotinamide, ultimately selecting a site located at a distance from the active site. Saturation mutations were performed on amino acids within the specified range and with high hydrophilicity. The distribution of mutation sites is as follows: Figure 1 As shown.

[0049] A library of randomized saturated mutants at 10 loci was constructed, and mutants in each library were identified using a nicotinic acid biosensor (NAsensor) screening platform. The nicotinamide mutant library was transformed into competent cells containing NAsensor. Using 30 mmol / L nicotinamide as a substrate and 0.1 mmol / L arabinose as an inducer, the cells were plated onto LB agar plates and incubated at 37°C for 16 h. Colonies with weaker fluorescence than the wild type were pre-screened in 96-well plates. The pre-screened strains were then transferred to 5 mL tubes for re-screening. The mutants exhibiting the most significant 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 mutant gene fragment was ligated into the vector pET28a(+), transformed into the BL21 expression host for induced expression, and its specific enzyme activity was measured. The results are as follows: Figure 2 As shown, a total of 16 mutants were obtained: F13Y, F13G, G55C, G55H, G55P, G55S, S59A, S59Y, S66I, S67A, S67E, S67L, A79S, Y99H, and R140V. Among them, 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 wild type. The superior mutant A79S was obtained, with a specific enzyme activity 4.74 times higher than that of WT (wild type).

[0050] 3. Enzymatic property analysis of WT and mutant nicotinamide enzymes:

[0051] We investigated the optimal temperature and temperature stability of WT and the mutant. Figure 3 As shown, the temperature detection range was 15-80℃. The catalytic activity of WT and mutants gradually increased with increasing temperature. The optimal temperature for WT and S67A was 37℃, and for A79S and S67A-A79S it was 40℃. With further increases in temperature, their enzyme activities decreased rapidly. The temperature stability differences between WT and mutants were explored by incubating at the corresponding temperatures for 20 min. The results are shown below. Figure 4 As shown, the mutant A79S exhibited improved stability at 50-60℃ compared to the wild type. Secondly, we investigated the optimal pH and pH stability of the WT and mutant, as shown... Figure 5 As shown, the pH detection range was 3.0-11.0. The catalytic activity of WT and the mutant gradually increased with increasing pH. The optimal pH for WT and S67A was 7.0, while the optimal temperature for A79S and S67A-A79S increased to 7.5 compared to the wild type. pH stability was measured after incubation at the corresponding pH for 20 min, and the results are shown in [Figure number missing]. Figure 6 The mutant A79S exhibits high stability within the pH range of 7.5-9.0, indicating improved alkali resistance.

[0052] We then determined the hydrophobicity of WT and mutant nicotinamide enzymes using the SDS-binding and bromophenol blue-binding methods. SDS was added to the protein solution, adjusted to 0.07 mM, and allowed to stand for 30 min. After dialyzing in phosphate buffer (pH 6.0) for 24 h, 0.5 ml of the dialysate was added to 10 ml of CHCl3, mixed thoroughly, and then 0.0024% methylene blue solution was added to the CHCl3 layer. After thorough mixing and centrifugation for 15 min, the bottom layer of SDS and methylene blue mixture was collected, and its absorbance was measured at 655 nm. The results are shown below. Figure 7 The hydrophobicity of mutants S67A, A79S, and S67A-A79S is 2.3 × 10⁻⁶. 5 2.2×10 5 2.1×10 5 Compared to WT's 1.87×10 5 The increases were 23%, 17.7%, and 12.3% higher, respectively, and were subsequently verified using the bromophenol blue binding method. Figure 8 The results were consistent with those obtained by the SDS binding assay, indicating that the enzyme activity of nicotinamide enzyme is positively correlated with hydrophobicity within a certain range. Meanwhile, we determined the kinetic parameters of WT and mutants under optimal conditions, as shown in Table 1. The Km values ​​of mutants S67A, A79S, and S67A-A79S were 5.1, 3.4, and 3.8 mM, respectively, lower than the 10.7 mM of WT, indicating increased affinity between the substrate and the mutants, with A79S showing the highest affinity. The Kcat values ​​of mutants S67A, A79S, and S67A-A79S were 10.9, 13.1, and 9.2 S, respectively. -1 The catalytic efficiencies of S67A, A79S, and S67A-A79S 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 nicotinic acid

[0056] The optimal temperature and pH for the culture of nitrile hydratase and nicotinamide enzyme mutants were determined using shake-flask catalysis. Wild-type strain catalysis, after six batches of feed, yielded 78.1 g / L NA and 68.3 g / L nicotinamide. Mutant strain A79S, after six batches of feed, yielded 143.3 g / L nicotinic acid with no accumulation of nicotinamide intermediates. The nicotinic acid yield of mutant strain A79S was 1.83 times that of wild-type, indicating that the catalytic activity of mutant strain A79S was significantly higher than that of wild-type. Subsequently, batch-feed catalysis with mixed strains was performed in a 1.5 L fermenter, with the following results: Figure 9 As shown, the catalytic rate decreased after the seventh batch of feed, and a total of ten batches were fed, ultimately yielding 224 g / L NA at a catalytic rate of 12.43 g / h / g dcw, with 13.3 g / L nicotinamide accumulation. To reduce the inhibition of 3-CN on nitrile hydratase and nicotinamide enzyme mutants, we employed a cascade fermentation method to produce NA. The cascade continuous catalytic reaction was used, and the cascade catalytic process is as follows: Figure 10 As shown, the reactor consists of two reactors: reactor 1 contains a nitrile hydratase strain, and reactor 2 contains a nicotinamide enzyme mutant strain (AMase mutant strain). They are connected by a hollow fiber column 3, forming a cascade system. A peristaltic pump 6, installed on the connecting pipe between reactor 1 and the feeding device 8, feeds the 3-CN at a rate of 15 mL / min to maintain a low concentration below 15 g / L. Concentration changes are monitored by HPLC. After 40 min, when 120 g / L of nicotinamide has accumulated in reactor 1, a second peristaltic pump 5, installed on the pipe between reactor 1 and reactor 2, is activated to feed the reaction solution from reactor 1 to reactor 2 containing the nicotinamide enzyme mutant at a rate of 40 mL / min. Meanwhile, the nitrile hydratase cells in the reaction solution of reactor 1 are refluxed back to reactor 1 through the hollow fiber column 3 on the pipe between reactor 1 and reactor 2. To prevent a decrease in the liquid volume in reactor 1, a phosphate buffer feeding bottle is connected to reactor 1 to maintain its volume at 1.1 L. After 20 minutes, 73 g / L NA 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, eventually stopping after 2.5 hours, yielding approximately 298.4 g / L NA. Figure 11 The yield was increased by 33.2% compared to the fed-batch method. Dividing the total yield by the total time yielded a rate of approximately 85.26 g / L / h for the conversion of 3-CN to NA. The cell dry weight was measured to be approximately 3.4 mg dcw / mL. Dividing the rate by the cell dry weight yielded a catalytic efficiency of 25.46 g / h / g dcw, representing a 104% improvement over the fed-batch method.

[0058] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A nicotinamide enzyme mutant, wherein the nucleotide sequence of a wild-type nicotinamide enzyme gene is shown in SEQ ID NO. 1, the nicotinamide enzyme mutant is that the alanine at position 79 of the nicotinamide enzyme with 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 of claim 1, wherein the nucleotide sequence of the gene is shown in SEQ ID NO.

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

5. The recombinant cell of claim 4, wherein The pET-28a(+) plasmid is used as a connection plasmid, and E. coli is used as an expression host. 6.Use of the mutant of claim 1, the gene of claim 2, the recombinant plasmid of claim 3, or the recombinant cell of claim 4 or 5 in the preparation of nicotinic acid.

7. A method of increasing the production of niacin, comprising, The alanine at position 79 of the nicotinamide enzyme with the amino acid sequence shown in SEQ ID NO. 2 is mutated to serine.

8. A method of increasing the production of niacin, comprising, The nitrile hydratase strain and the nicotinamide enzyme mutant strain are used for cascade continuous catalysis, the gene sequence of the nitrile hydratase in the nitrile hydratase strain is shown in SEQ ID NO. 5, and the gene sequence of the nicotinamide enzyme mutant in the nicotinamide enzyme mutant strain is shown in SEQ ID NO.

3.

9. The method of claim 8, wherein, The nitrile hydratase strain is used for catalysis in a first reactor, and the nicotinamide enzyme mutant strain is used for catalysis in a second reactor; the two reactors are connected to form a cascade system by using a hollow fiber column and a peristaltic pump; when the nicotinamide accumulates to a certain amount in the first reactor, the reaction solution in the first reactor is pumped into the second reactor through the hollow fiber column; after a suitable reaction time, the nicotinic acid clear solution is continuously filtered out from the second reactor.

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

Images