Nitrile hydratase mutant as well as production method and application thereof

Abstract

The invention provides a nitrile hydratase mutant as well as a production method and application thereof, and belongs to the technical field of biological enzymes. The invention provides a nitrile hydratase mutant. The nitrile hydratase mutant comprises an amino acid sequence as shown in SEQ ID No. 1. The invention further provides a production method of the nitrile hydratase mutant, large-scale production is carried out through microbial fermentation, the reaction condition is mild, the operation process is simple and convenient, and the method is suitable for industrial production. The nitrile hydratase mutant can be used as a catalyst, a thallus containing the nitrile hydratase mutant can be used as a catalyst, a crushing liquid of the thallus can be used as a catalyst to catalyze nicotinamide biosynthesis of nicotinamide, and in a catalytic system, the highest adding amount of nicotinamide can reach 400 g / L, and high-purity nicotinamide can be efficiently generated.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioenzymes, and particularly relates to a nitrile hydratase mutant, a production method and an application thereof. Background Art

[0002] Nicotinamide (CAS No.: 98-92-0), also known as niacinamide, vitamin B3 or vitamin PP, is a water-soluble vitamin belonging to the B vitamin family and is a component of coenzyme I (nicotinamide adenine dinucleotide, NAD) and coenzyme II (nicotinamide adenine dinucleotide phosphate, NADP). In the human body, the nicotinamide part in the structures of these two coenzymes has reversible hydrogenation and dehydrogenation characteristics, plays a role in transferring hydrogen in biological oxidation, can promote tissue respiration, biological oxidation processes and metabolism, and is of great significance for maintaining the integrity of normal tissues, especially the skin, digestive tract and nervous system.

[0003] At present, the application of nicotinamide in whitening is becoming more and more extensive, but the production mode of nicotinamide is difficult to meet the demand for nicotinamide. The synthesis of nicotinamide mainly includes chemical synthesis and biocatalysis. Chemical synthesis requires reactions under high temperature and high pressure conditions and is prone to produce toxic by-products, etc. Biocatalysis often uses nitrile hydratase. However, in actual production, the following problems will occur, thus increasing the cost: a certain proportion of organic co-solvent often needs to be added to the catalytic system. Although it can increase the solubility of raw materials to a certain extent, it will increase the cost and reduce the reuse rate of cells; phosphates or other components are often used as the main raw materials of the buffer solution in the catalytic system, increasing the subsequent extraction steps; the catalyst in biocatalysis is often disposable and requires single fermentation and single catalysis, increasing the production cost.

[0004] Therefore, in view of the above difficulties, there is an urgent need for a biocatalyst with high catalytic activity, high reuse rate and independent of buffer salts to catalyze the synthesis of nicotinamide from 3-cyanopyridine to meet the requirements of industrial production. Summary of the Invention

[0005] The present invention provides a nitrile hydratase mutant, a production method and an application thereof. The nitrile hydratase mutant is a water-soluble protein, can be mass-produced by microbial fermentation, has mild reaction conditions and a simple operation process, and is suitable for industrial production.

[0006] The present invention provides a nitrile hydratase mutant, including any one of the following amino acid sequences:

[0007] (1) The amino acid sequence shown in SEQ ID No.1;

[0008] (2) An amino acid sequence having more than 85% homology with the amino acid sequence shown in SEQ ID No.1 and formed after one or more mutations, additions and / or deletions of the amino acid sequence.

[0009] The present invention also provides a nucleic acid molecule encoding the above-mentioned nitrile hydratase mutant.

[0010] In a preferred embodiment of the present invention, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID No.2.

[0011] The present invention also provides a recombinant plasmid containing the above-mentioned nucleic acid molecule.

[0012] The present invention also provides a recombinant bacterium containing the above-mentioned nucleic acid molecule or the above-mentioned recombinant plasmid.

[0013] The present invention also provides a method for producing the above-mentioned nitrile hydratase mutant, which includes connecting the above-mentioned nucleic acid molecule with a basic vector to construct a recombinant plasmid, transforming a host bacterium with the recombinant plasmid to construct a recombinant bacterium;

[0014] Culturing the recombinant bacterium, and the nitrile hydratase mutant is contained in the bacterial cells.

[0015] The present invention also provides a reagent containing the nitrile hydratase mutant, which includes wet bacterial cells obtained by fermenting and culturing the above-mentioned recombinant bacterium, or crude enzyme solution obtained by disrupting the wet bacterial cells, or pure enzyme solution obtained by purifying the crude enzyme solution, or the above-mentioned nitrile hydratase mutant.

[0016] The present invention also provides a biocatalytic method for synthesizing nicotinamide, which includes using the above-mentioned reagent as a catalyst, using nicotinonitrile as a substrate, and synthesizing nicotinamide through a biocatalytic reaction.

[0017] In a preferred embodiment of the present invention, the addition amount of the catalyst is 5-50 g / L of the catalytic system, and during the biocatalytic reaction process, nicotinonitrile is added by a batch feeding method, and the batch feeding speed is 50-200 g / h.

[0018] In a preferred embodiment of the present invention, the biocatalytic reaction is accompanied by shaking, the shaking frequency is 100-600 rpm, the temperature is 25-60 °C, and the reaction time is 3-12 h.

[0019] Beneficial effects: The present invention provides a nitrile hydratase mutant, which includes the amino acid sequence shown in SEQ ID No.1. The nitrile hydratase mutant of the present invention is a water-soluble protein and can be solubly expressed in the cell supernatant, so it can be mass-produced through an expression system. The present invention also provides a production method of the nitrile hydratase mutant, which is mass-produced through microbial fermentation, the reaction conditions are mild, the operation process is simple, it is suitable for industrial production, and solves the technical problems in the prior art that large-scale production cannot be carried out or the cost is high due to the need for organic cosolvents and salt ion buffers, etc.

[0020] The present invention can use the nitrile hydratase mutant as a catalyst, or use the bacterial cells containing the nitrile hydratase mutant as a catalyst, or use the lysate of the bacterial cells as a catalyst to catalyze the biosynthesis of nicotinamide from 3-cyanopyridine. In the catalytic system, the maximum addition amount of 3-cyanopyridine can reach 400 g / L, and high-purity nicotinamide can be efficiently generated. Detailed implementation manners

[0021] The present invention provides a nitrile hydratase mutant, including the amino acid sequence of any one of the following:

[0022] (1) The amino acid sequence shown in SEQ ID No.1;

[0023] (2) An amino acid sequence having a homology of more than 85% with the amino acid sequence shown in SEQ ID No.1, and formed after one or more mutations, additions, and / or deletions of the amino acid sequence.

[0024] The amino acid sequence of the nitrile hydratase mutant of the present invention can be the one shown in SEQ ID No.1, or a homologous sequence of the sequence shown in SEQ ID No.1. Even one or more amino acid mutations, additions, or deletions can occur on the basis of the homologous sequence, as long as the basic function of the nitrile hydratase mutant can be maintained, it belongs to the protection scope of the present field.

[0025] In one embodiment of the present invention, taking SEQ ID No.1 as an example for illustration, wherein SEQ ID No.1: MTRVHDRLGRFGDGPVTAEAEDVVFQTDDHRRALALTLATGTLRAWNIDVSRHARECLSEKWLAALTDLLVAQGVLSRADLSAASDAPDTIHPLAARHLPAARVAAVLAAGGPADRDSTVAARYQPGDIVRTRLSGNRSVAGGHTRLPTVPGGHTRLPAYANPVTHTRLPRYARAKTGVVEAVQGAFVFPDDNAHGEGENPQWLYTVVFDGAELWGGDADPTLTVSIWAHPEHPRDEVVLDLWQTYLVHP.

[0026] The present invention also provides a nucleic acid molecule encoding the above-mentioned nitrile hydratase mutant.

[0027] The nucleic acid molecule of the present invention can be ribonucleotides and / or deoxyribonucleotides, and its forms include single-stranded DNA or RNA, double-stranded DNA or RNA, multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemical or biochemical modified, unnatural or derivative nucleobases. In a preferred embodiment of the present invention, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID No.2:

[0028] ATGACTCGCGTTCATGATCGTCTGGGTCGTTTCGGCGATGGTCCGGTTACT

[0029] GCAGAAGCAGAGGACGTGGTTTTCCAGACCGACGACCACCGTCGCGCCCT

[0030] GGCGCTGACTCTGGCAACTGGCACCCTGCGTGCTTGGAACATCGACGTCT

[0031] CTCGTCACGCCCGTGAGTGCCTGAGCGAAAAATGGCTGGCAGCCCTGACC

[0032] GATCTGCTGGTAGCACAAGGCGTTCTGTCCCGTGCAGATCTGTCCGCGGCA

[0033] TCTGATGCGCCGGACACCATCCACCCACTGGCGGCGCGTCATCTGCCGGC

[0034] GGCCCGTGTTGCTGCTGTACTGGCTGCTGGTGGTCCAGCCGACCGTGACT

[0035] CTACCGTGGCAGCTCGTTATCAGCCAGGTGACATCGTTCGTACTCGTCTGT

[0036] CCGGTAACCGTTCTGTGGCGGGCGGCCACACCCGTCTGCCGACTGTCCCA

[0037] GGTGGTCATACTCGCCTGCCGGCTTATGCAAACCCGGTCACGCACACCCGT

[0038] CTGCCGCGTTACGCACGTGCGAAAACCGGCGTCGTGGAAGCTGTACAGGG

[0039] TGCCTTCGTATTCCCGGACGATAACGCCCACGGCGAAGGTGAGAACCCGC

[0040] AGTGGCTGTACACCGTAGTTTTCGATGGCGCCGAGCTGTGGGGTGGTGATG

[0041] CCGACCCGACCCTGACCGTAAGCATCTGGGCGCATCCTGAACATCCGCGCGATGAAGTGGTTCTGGACCTGTGGCAGACCTACCTGGTACATCCGTAA。

[0042] The present invention also provides a recombinant plasmid containing the above nucleic acid molecule.

[0043] The recombinant plasmid described in the present invention is mainly based on vectors commonly used in the art for expression systems, such as vectors for prokaryotic expression and vectors for eukaryotic expression. Specifically, it can be pET-21a(+), pET-28a(+), pET-22b(+), pETDuet-1 or pACYCDuet-1. In one embodiment, pET-22b(+) is taken as an example for illustration, but it cannot be regarded as the entire protection scope of the present invention.

[0044] In the embodiment of the present invention, the sequence shown in SEQ ID No.2 is inserted between NdeI and XhoI of pET-22b(+) to construct the recombinant plasmid.

[0045] The present invention also provides a recombinant bacterium containing the above nucleic acid molecule or the above recombinant plasmid.

[0046] The recombinant bacterium described in the present invention can use bacteria or fungi as host bacteria, such as Escherichia coli, Bacillus subtilis or yeast as host bacteria. Specifically, such as Escherichia coli E.coli BE21(DE3), Bacillus subtilis BL21(DE3)pLysS or Saccharomyces cerevisiae Rosetta(DE3).

[0047] The present invention also provides a method for producing the above-mentioned nitrile hydratase mutant, including connecting the above nucleic acid molecule with a basic vector to construct a recombinant plasmid, transforming a host bacterium with the recombinant plasmid to construct a recombinant bacterium;

[0048] Culturing the recombinant bacterium, and the nitrile hydratase mutant is contained in the bacterial cells.

[0049] In one embodiment of the present invention, the above recombinant plasmid was transformed into Escherichia coli E. coli BE21(DE3) to construct an engineered strain E. coli BL21(DE3) / pET-22b(+)-NHasemut.

[0050] The present invention uses the engineered strain E. coli BL21(DE3) / pET-22b(+)-NHasemut as the production strain, and ferments and cultures the production strain, such as culturing in an LB liquid medium containing ampicillin until the OD 600 reaches 1.5 to 2.0, then adding IPTG for induction. During the induction process, the temperature is slightly lower than the temperature during the culture. For example, fermentation is carried out at 30 °C, and the glucose concentration in the tank is controlled at ≤0.5 g / L by feeding an 80% glucose solution. The total fermentation time is 15 to 24 h, or when the OD reaches about 100, the fermentation is stopped to harvest the bacteria, and the harvested bacteria contain the nitrile hydratase mutant.

[0051] The present invention also provides a reagent containing a nitrile hydratase mutant, including wet bacteria obtained after fermenting and culturing the above recombinant bacteria, or a crude enzyme solution obtained by disrupting the wet bacteria, or a pure enzyme solution obtained by purifying the crude enzyme solution, or the above nitrile hydratase mutant.

[0052] The present invention can use the wet bacteria after culturing and fermentation as a catalyst, or use the crude enzyme solution obtained by disrupting the wet bacteria as a catalyst, or centrifuge the crude enzyme solution and use the supernatant as a catalyst, or even purify the crude enzyme solution and use the pure enzyme solution as a catalyst. Of course, it can also directly use the nitrile hydratase mutant generated by other methods as a catalyst.

[0053] The present invention also provides a biocatalytic method for synthesizing nicotinamide, including using the above reagent as a catalyst and using nicotinonitrile as a substrate to synthesize nicotinamide through a biocatalytic reaction.

[0054] In one embodiment of the present invention, using the above-mentioned wet bacterial cells as a catalyst, before use, the wet bacterial cells can be mixed with a buffer solution or water. The buffer solution can be a phosphate buffer solution with a pH value of 6.5 - 8.0. In the catalytic system, when the catalyst is wet bacterial cells, the dosage of the catalyst is 5 - 50 g of wet bacterial cells / L of the catalytic system. When performing the catalysis in the present invention, the 3-cyanopyridine is added in a batchwise feeding manner, and the batchwise feeding rate is 50 - 200 g / h, such as 50 g / h, 60 g / h, 70 g / h, 80 g / h, 90 g / h, 100 g / h, 110 g / h, 120 g / h, 130 g / h, 140 g / h, 150 g / h, 160 g / h, 170 g / h, 180 g / h, 190 g / h or 200 g / h. In one embodiment, it is added at a rate of 100 g / h.

[0055] The catalytic reaction of the present invention is carried out under shaking conditions, and the shaking frequency is 100 - 600 rpm, such as 100 rpm, 200 rpm, 300 rpm, 400 rpm, 500 rpm or 600 rpm. In one embodiment, a shaking rate of 200 rpm is selected for shaking. The temperature of the catalytic reaction of the present invention is 25 - 60 °C, such as it can be 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45 °C, 46 °C, 47 °C, 48 °C, 49 °C, 50 °C, 51 °C, 52 °C, 53 °C, 54 °C, 55 °C, 56 °C, 57 °C, 58 °C, 59 °C or 60 °C. In one embodiment, 30 °C is selected for the catalytic reaction.

[0056] The time of the catalytic reaction of the present invention is 3 - 12 h, such as it can be 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h or 12 h. After the catalytic reaction of the present invention is completed, it further includes extracting and purifying the reaction solution.

[0057] In the present invention, the listed numbers are only exemplary data that can meet the experimental conditions. Of course, they can also be non-integer data in the form of decimals or fractions between any two numbers.

[0058] To further illustrate the present invention, the following examples are used to describe in detail a nitrile hydratase mutant and its production method and application provided by the present invention, but they should not be construed as limiting the protection scope of the present invention.

[0059] In the embodiments of the present invention, unless otherwise specified, the materials used are prepared according to existing methods or directly purchased from the market.

[0060] Composition of the seed medium: 20 g / L of glucose, 1.5 g / L of yeast extract powder, 1.2 g / L of peptone, 1.5 g / L of (NH4)2SO4, 1.2 g / L of K2HPO4, 0.8 g / L of MgSO4, 1 g / L of citric acid, 10 mg / L of MnSO4·H2O, and 8 mg / L of FeSO4·7H2O.

[0061] Composition of the fermentation medium: 1.5 g / L of disodium hydrogen phosphate anhydrous, 1.8 g / L of potassium dihydrogen phosphate, 0.7 g / L of ammonium chloride, 0.4 g / L of anhydrous magnesium sulfate, 120 g / L of glucose, 4 g / L of peptone, 2.4 g / L of yeast extract powder, 0.8 g / L of anhydrous sodium sulfate, 30 mg / L of manganese chloride tetrahydrate, 20 mg / L of ferrous sulfate, 1 g / L of citric acid, 2 mg / L of vitamin mixture and biotin. The vitamin mixture includes vitamin B1, vitamin B3, vitamin B5, and vitamin B12.

[0062] Example 1: Construction of the recombinant expression plasmid pET-22b(+)-NHasemut

[0063] Entrusted Suzhou Genewiz Biotechnology Co., Ltd. to synthesize the nucleotide sequence of the nitrile hydratase mutant shown in SEQ ID NO.2.

[0064] pET-22b(+) was digested with the restriction enzymes NdeI and XhoI (Thermo Fisher Scientific). Seamless cloning primers were designed according to the nucleotide sequence of the nitrile hydratase mutant.

[0065] Forward primer (SEQ ID No.3): TGTTTAACTTTAAGAAGGAGATATACATATGGATGGCATTCACGACAC;

[0066] Reverse primer (SEQ ID No.4): TGTCGACGGAGCTCGAATTCGGATCCTTAATCAATAATCGCCATGCTT.

[0067] The reaction system for the PCR amplification reaction was: 2×PhantaMax Buffer 25 μL, dNTP Mix (10 mM each) 1 μL, 10 μM forward primer 2 μL, 10 μM reverse primer 2 μL, template 1 μL, Phanta Max Super-Fidelity DNA Polymerase 1 μL, and ddH2O was added to make the total volume 50 μL.

[0068] The reaction procedure of the PCR amplification reaction was as follows: pre-denaturation at 98°C for 2 minutes; denaturation at 98°C for 20 seconds, annealing at 56°C for 20 seconds, extension at 72°C for 5 minutes, for 30 cycles; extension at 72°C for 10 minutes.

[0069] The plasmid long fragment and the PCR product were recovered using an agarose gel DNA recovery kit (DP209-02, TIANGEN). Using the seamless cloning kit (C115-02, Vazyme) from Novizan, the PCR purified product and the cut pET-22b(+) long fragment were subjected to seamless ligation.

[0070] The ligation system was as follows: 2 μL of plasmid long fragment, 1 μL of target gene fragment, 1 μL of 10× Ligase Buffer, and ddH2O was added to make up the total volume to 10 μL.

[0071] The ligation procedure: ligation at 50°C for 30 minutes.

[0072] The ligation product was transformed into competent cells of Escherichia coli E. coli DH5α, and the recombinants were screened on an LB plate containing ampicillin resistance. Single colonies were picked for colony PCR verification.

[0073] The identification primers used were the universal primers T7 and T7 ter. The positive clones were sent to Genewiz Biotechnology Co., Ltd. for sequencing, and the positive clone pET-22b(+)-NHasemut was obtained.

[0074] Example 2: Construction of recombinant genetically engineered bacteria

[0075] The positive clone pET-22b(+)-NHasemut identified correctly in Example 1 was picked, and the engineered bacteria were constructed respectively according to the following steps:

[0076] The positive clone was inoculated into 5 mL of LB liquid medium containing ampicillin (10 g / L peptone, 5 g / L yeast extract powder, and 10 g / L NaCl, pH 7.0), cultured overnight at 37°C with 200 rpm. The recombinant expression plasmid was extracted using a plasmid miniprep kit, transformed into competent cells of Escherichia coli E. coli BL21(DE3), spread on an LB plate containing ampicillin, and cultured overnight at 37°C in an inverted position. Large colonies were picked and re-inoculated into an LB liquid medium containing ampicillin, and the bacterial strain was cryopreserved with glycerol at a total concentration of 20%, thus obtaining the engineered bacteria E. coli BL21(DE3) / pET-22b(+)-NHasemut.

[0077] Example 3: Fermentation and preparation of nitrile hydratase mutant

[0078] Using the engineered bacterium E. coli BL21(DE3) / pET-22b(+)-NHasemut constructed in Example 2, fermentation and expression were carried out according to the following steps:

[0079] Plate activation: Dip the glycerol stock solution with an inoculation loop and perform quadrant streaking on an LB solid plate containing 100 μg / mL ampicillin. Incubate it upside down in a 37°C constant temperature incubator overnight.

[0080] Tube activation: After the above plate activation, pick a large monoclonal colony and inoculate it into an LB liquid medium containing 100 μg / mL ampicillin for activation. Then streak it again on an LB solid plate containing 100 μg / mL ampicillin within one week.

[0081] Seed culture: Pick a large single colony from the solid plate and inoculate it into a seed medium containing 100 μg / mL ampicillin. The liquid loading volume is 30 mL / 250 mL. Incubate it on a shaker at 37°C and 200 rpm for 12 h to obtain the seed solution.

[0082] Fermentation culture: Inoculate the seed solution into a 10 L fermenter at an inoculation amount of 1%. The fermentation medium (6 L of fermentation medium containing 100 μg / mL ampicillin), the initial culture temperature is 37°C. Maintain the culture pH at 7.0 ± 0.2 by automatically adding aqueous ammonia solution, and maintain the dissolved oxygen value of the culture at 30 - 50% by adjusting the stirring speed or ventilation volume. When OD 600 reaches 15 - 20, add an inducer IPTG with a final concentration of 0.1 mM and lower the culture temperature to 30°C to start fermentation. Control the glucose concentration in the tank at ≤0.5 g / L by adding an 80% glucose solution. The total fermentation duration is 15 - 24 h, or when OD reaches about 100, harvest the bacteria by discharging the tank to obtain the Nhasemut catalyst. Resuspend it with water according to the wet cell mass fraction of 1% to obtain the catalytic system.

[0083] Example 4: Catalysis of 3-cyanopyridine to nicotinamide by the nitrile hydratase mutant

[0084] In this example, the Nhasemut whole cells prepared in Example 3 were used as the catalyst to catalyze the formation of nicotinamide from 3-cyanopyridine. The specific operation is as follows: Resuspend it with water according to the wet cell mass fraction of 1% to obtain the catalytic system. The batch feeding rate of 3-cyanopyridine is 100 g / h. When the substrate addition amount reaches 400 g / L, stop the feeding and continue the reaction until the raw materials are completely converted, then stop the reaction. At the same time, Table 1 compares the effects of batch feeding and one-time addition of raw materials on the product concentration. The results show that batch feeding can significantly increase the product concentration.

[0085] After the catalysis is completed, the fermentation broth is subjected to extraction and purification. Add an equal volume of methanol to dilute and terminate the system, perform suction filtration, remove the solvent by vacuum distillation, and use methanol to carry twice to completely remove the aqueous phase. The crude product is dissolved by heating with 3V methanol, the insoluble matter is filtered off, the filtrate is cooled, solids start to precipitate at 29 °C, the temperature is cooled to 10 °C, suction filtration is performed, and the filter cake wet product is obtained by rinsing with cold methanol. The wet product is vacuum dried at 60 °C to obtain the product, which is further pulverized. The content and purity of nicotinamide detected by liquid phase are both greater than 99.5%.

[0086] Table 1 Influence of fed-batch and one-time addition of raw materials on product concentration

[0087] One-time addition Fed-Batch Niacinamide concentration 463.8g / L 231.4g / L

[0088] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments according to these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A nitrile hydratase mutant, characterized in that, An amino acid sequence comprising any one of the following: (1) The amino acid sequence shown in SEQ ID No.1; (2) An amino acid sequence having a homology of more than 85% with the amino acid sequence shown in SEQ ID No.1 and formed after one or more amino acid sequence mutations, additions and / or deletions.

2. A nucleic acid molecule encoding the nitrile hydratase mutant according to claim 1.

3. The nucleic acid molecule according to claim 2, wherein The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID No.

2.

4. A recombinant plasmid comprising the nucleic acid molecule according to claim 2 or 3.

5. A recombinant bacterium containing the nucleic acid molecule according to claim 2 or 3 or the recombinant plasmid according to claim 4.

6. A method for producing the nitrile hydratase mutant according to claim 1, characterized in that, It includes connecting the nucleic acid molecule according to claim 2 or 3 with a basic vector to construct a recombinant plasmid, and transforming a host bacterium with the recombinant plasmid to construct a recombinant bacterium; Culturing the recombinant bacterium, and the nitrile hydratase mutant is contained in the bacterial cells.

7. A reagent containing a nitrile hydratase mutant, characterized in that, It includes wet bacterial cells obtained after fermentation culture of the recombinant bacterium according to claim 5, or a crude enzyme solution obtained after crushing the wet bacterial cells, or a pure enzyme solution obtained after purification of the crude enzyme solution, or the nitrile hydratase mutant according to claim 1.

8. A biocatalytic method for synthesizing nicotinamide, characterized in that, It includes synthesizing nicotinamide through a biocatalytic reaction using the reagent according to claim 7 as a catalyst and using nicotinonitrile as a substrate.

9. The biocatalytic method according to claim 8, wherein The addition amount of the catalyst is 5 - 50 g / L of the catalytic system, and during the biocatalytic reaction process, nicotinonitrile is added by a method of batch feeding, and the batch feeding rate is 50 - 200 g / h.

10. The biocatalytic method according to claim 8 or 9, characterized in that, During the biocatalytic reaction, shaking is accompanied, the shaking frequency is 100 - 600 rpm, the temperature is 25 - 60 °C, and the reaction time is 3 - 12 h.