A nitrile hydratase mutant and its application

Abstract

The present invention belongs to the technical field of enzyme engineering, and in particular relates to a nitrile hydratase mutant and its application, wherein the nitrile hydratase mutant is obtained by performing multi-point mutations at multiple sites in the 98th position of the amino acid sequence shown in SEQ ID NO.1 and the 41st, 51st and 55th positions of the amino acid sequence shown in SEQ ID NO.2. The present invention performs iterative saturation mutation on nitrile hydratase, and provides multiple nitrile hydratase mutants with significantly improved product tolerance, and these mutants can be used to produce a variety of amide compounds, and compared with the starting type, the specific enzyme activity and catalytic performance are significantly improved.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and in particular relates to a nitrile hydratase mutant and application thereof. Background Art

[0002] Amide compounds have high polarity, high stability and structural diversity. They are widely present in drugs and bioactive molecules and are also important raw materials for organic synthesis. They are widely used in drug synthesis, agriculture and fine chemical industries and are in great demand. The green and efficient synthesis of amide compounds has always been a research hotspot in the industry.

[0003] In traditional chemical methods, the most common amide synthesis route is to couple active carboxylic acids or their derivatives with amines to generate corresponding amides under the action of coupling reagents. The safety and atom economy of this process are poor, and the obtained amide compounds also require cumbersome separation and purification steps, and the process economy is poor. The bioenzymatic method with nitrile hydratase (NHase) as the core is widely used in catalyzing the hydration of nitrile compounds to generate amide substances. Biocatalysis is recognized as a green catalysis due to its mild reaction conditions, high atom economy, and environmental friendliness. The hydration of nitrile substances with nitrile hydratase as the core to generate corresponding amide compounds is a typical bioenzyme catalyzed reaction. The bioenzymatic production of acrylamide and nicotinamide has become the main method for the industrial production of these two amide compounds. At present, the annual production capacity of acrylamide by bio-method in the world has reached nearly one million tons, and the annual production capacity of nicotinamide has also reached nearly 100,000 tons.

[0004] When catalyzing the hydration reaction of nitrile compounds, the strong toxicity of the substrate and the product often damages the enzyme molecules, causing them to be irreversibly and rapidly inactivated. The high concentration of acrylamide that accumulates continuously during the reaction inhibits nitrile hydratase, resulting in the concentration of acrylamide produced by industrial biocatalysis in China being generally only about 30%. Therefore, improving the product tolerance of nitrile hydratase and increasing the product concentration to improve the economic efficiency of the process has always been pursued by the industry.

[0005] By constructing a genetically engineered strain with nitrile hydratase catalytic activity and carrying out protein engineering transformation on nitrile hydratase, the practical problems of amide compounds in production can be solved to a certain extent. Patent document (CN107881163A) discloses a nitrile hydratase (NHAM) gene sequence derived from Aurantimonas manganoxydans SI859A, which has high catalytic activity for acrylonitrile and good thermal stability. Patent document (CN116162612) carries out protein engineering transformation on NHAM, and a single-point mutant of nitrile hydratase with significantly improved product tolerance and no loss of enzyme activity can be obtained. Through iterative saturation mutation, the present invention further obtains a multi-point mutant with more excellent product tolerance and significantly improved specific enzyme activity. Summary of the invention

[0006] In view of the need for nitrile hydratase with higher tolerance and enzyme activity in the prior art, the present invention performs iterative saturation mutation on four sites (α-R98, β-G41, β-S51, β-R55) of α and β subunits of nitrile hydratase (NHAM) derived from Aurantimonas manganoxydans SI859A, and provides multiple nitrile hydratase mutants with significantly improved product tolerance and their catalytic effects on multiple substrates.

[0007] In a first aspect, the present invention provides a nitrile hydratase mutant, wherein the nitrile hydratase mutant is obtained by mutating one or more of the 98th position of the amino acid sequence shown in SEQ ID NO.1, the 41st position, the 51st position and the 55th position of the amino acid sequence shown in SEQ ID NO.2.

[0008] The nitrile hydratase mutant is any one of the following:

[0009] (1) mutating the 98th arginine of the α subunit of the nitrile hydratase whose amino acid sequence is shown in SEQ ID NO.1 to isoleucine, and mutating the 41st glycine of the β subunit of the nitrile hydratase whose amino acid sequence is shown in SEQ ID NO.2 to aspartic acid;

[0010] (2) mutating the 98th arginine of the α subunit of the nitrile hydratase whose amino acid sequence is shown in SEQ ID NO.1 to valine, and mutating the 41st glycine of the β subunit of the nitrile hydratase whose amino acid sequence is shown in SEQ ID NO.2 to aspartic acid;

[0011] (3) the serine at position 51 of the β subunit of the nitrile hydratase whose amino acid sequence is shown in SEQ ID NO.2 is mutated to glutamic acid, and the arginine at position 98 of the α subunit is mutated to valine;

[0012] (4) the serine at position 51 of the β subunit of the nitrile hydratase whose amino acid sequence is shown in SEQ ID NO.2 is mutated to glutamic acid, and the arginine at position 98 of the α subunit is mutated to isoleucine;

[0013] (5) mutating the arginine at position 98 of the α subunit of the nitrile hydratase with an amino acid sequence such as that shown in SEQ ID NO.1 to valine, and mutating the arginine at position 55 of the β subunit of the nitrile hydratase with an amino acid sequence such as that shown in SEQ ID NO.2 to cysteine;

[0014] (6) The serine at position 51 of the β subunit of the nitrile hydratase whose amino acid sequence is shown in SEQ ID NO.2 is mutated to tryptophan, and the arginine at position 55 is mutated to cysteine;

[0015] (7) The glycine at position 41 of the β subunit of the nitrile hydratase whose amino acid sequence is shown in SEQ ID NO.2 is mutated to aspartic acid, the serine at position 51 is mutated to tryptophan, and the arginine at position 55 is mutated to cysteine;

[0016] (8) The glycine at position 41 of the β subunit of the nitrile hydratase whose amino acid sequence is shown in SEQ ID NO.2 is mutated to serine, the serine at position 51 is mutated to tryptophan, and the arginine at position 55 is mutated to cysteine;

[0017] (9) mutating the arginine at position 98 of the α subunit of the nitrile hydratase with an amino acid sequence such as that shown in SEQ ID NO.1 to isoleucine, and mutating the glycine at position 41 of the β subunit of the nitrile hydratase with an amino acid sequence such as that shown in SEQ ID NO.2 to aspartic acid, and mutating the serine at position 51 to valine;

[0018] (10) mutating the arginine at position 98 of the α subunit of the nitrile hydratase with an amino acid sequence as shown in SEQ ID NO.1 to valine, and mutating the arginine at position 55 of the β subunit of the nitrile hydratase with an amino acid sequence as shown in SEQ ID NO.2 to cysteine, and mutating the serine at position 51 to asparagine;

[0019] (11) mutating the arginine at position 98 of the α subunit of the nitrile hydratase with an amino acid sequence as shown in SEQ ID NO.1 to methionine, and mutating the glycine at position 41 of the β subunit of the nitrile hydratase with an amino acid sequence as shown in SEQ ID NO.2 to aspartic acid, the serine at position 51 to tryptophan, and the arginine at position 55 to cysteine;

[0020] (12) mutating the arginine at position 98 of the α subunit of the nitrile hydratase with an amino acid sequence such as SEQ ID NO.1 to isoleucine, and mutating the glycine at position 41 of the β subunit of the nitrile hydratase with an amino acid sequence such as SEQ ID NO.2 to aspartic acid, the serine at position 51 to tryptophan, and the arginine at position 55 to cysteine;

[0021] (13) The arginine at position 98 of the α subunit of the nitrile hydratase with an amino acid sequence as shown in SEQ ID NO.1 is mutated to leucine, and the glycine at position 41 of the β subunit of the nitrile hydratase with an amino acid sequence as shown in SEQ ID NO.2 is mutated to aspartic acid, the serine at position 51 is mutated to tryptophan, and the arginine at position 55 is mutated to cysteine.

[0022] The nitrile hydratase NHAM from Aurantimonas manganoxydans SI859A consists of an α subunit, a β subunit and a regulatory protein, and its amino acid sequence is shown in SEQ ID NO.1, NO.2 and NO.3. Saturation iterative mutation is performed on it to obtain the above mutant.

[0023] In a second aspect, the present invention provides a gene encoding the above-mentioned nitrile hydratase mutant.

[0024] In a third aspect, the present invention provides a recombinant expression vector comprising the gene as claimed in claim 3.

[0025] In a fourth aspect, the present invention provides a genetically engineered bacterium, wherein a host cell of the genetically engineered bacterium comprises the gene as claimed in claim 3, or comprises the recombinant expression vector as claimed in claim 4.

[0026] The gene encoding the β subunit mutation described in the first aspect (SEQ ID No. 5) or the gene encoding the α subunit mutation (SEQ ID No. 4) is combined with the regulatory protein gene (SEQ ID No. 6) to obtain the nitrile hydratase gene cluster α-β-p14k, and the connection sequence between the β subunit and the regulatory protein p14k is shown in SEQ ID NO. 7; the gene cluster is constructed into the expression vector pET-28a (+), and transferred into the genetically engineered Escherichia coli E. coli BL21 (DE3) to obtain a mutant strain.

[0027] In a fifth aspect, the present invention provides an enzyme preparation comprising the mutant as described in claim 1 or 2.

[0028] In a sixth aspect, the present invention provides the use of the nitrile hydratase mutant, or the genetically engineered bacteria, or the enzyme preparation in catalyzing the production of amide substances from aliphatic or aromatic nitrile compounds, wherein the aliphatic nitrile compounds include acrylonitrile, isobutyronitrile, adiponitrile, and 2-butenenitrile, and the aromatic nitrile compounds include 3-cyanopyridine, 4-cyanopyridine, benzonitrile, and 2,6-difluorobenzonitrile.

[0029] The present invention has the following beneficial effects:

[0030] The invention significantly improves the stable expression of nitrile hydratase NHAM from Aurantimonas manganoxydans SI859A in genetically engineered bacteria E. coli BL21 (DE3) through iterative saturation mutation. With acrylonitrile as a substrate, the tolerance of the nitrile hydratase mutant in 500 g / L acrylamide solution is significantly improved, and the mutant has a wide substrate spectrum, and the mutant has significantly improved catalytic activity to a variety of nitrile compounds, and has a very large industrial application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 These are the initial relative enzyme activities of the initial WT and mutant forms of nitrile hydratase and the residual enzyme activity after immersion in 500 g / L acrylamide solution for 30 minutes.

[0032] Figure 2 The figure shows the results of substrate spectrum determination of the initial WT and mutant forms of nitrile hydratase. DETAILED DESCRIPTION

[0033] The experimental methods in the present invention are all conventional methods unless otherwise specified. For details of gene cloning operations, please refer to "Molecular Cloning Experiment Guide" compiled by J. Sambrook et al.

[0034] The recombinant Escherichia coli with nitrile hydratase gene involved in the present invention uses pET-28a(+) as the vector and Escherichia coli BL21(DE3) as the host. The kit for preparing the competent cells was purchased from TAKARA.

[0035] Reagents used in the downstream catalytic process: acrylonitrile was purchased from Sinopharm Chemical Reagent Co., Ltd.; acetamide was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; acrylamide was purchased from Aladdin Reagent Co., Ltd.; other commonly used reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. The three-letter or single-letter representation of amino acids used in this application text adopts the amino acid code specified by IUPAC (Eur. J. Biochem., 138: 9-37, 1984).

[0036] Definition of enzyme activity (U / mL): the amount of enzyme required to catalyze the substrate acrylonitrile to produce 1 μmol acrylamide per minute.

[0037] Standard detection system for nitrile hydratase activity: appropriate amount of enzyme solution, 4mM substrate, total system of 500μL, reaction medium of 0.25M phosphate buffer at pH 7.5. Reaction at 28℃ for 5min, centrifuge the reaction solution at 12000rpm for 3min, take the supernatant and dilute it 10 times, pass it through a 0.22μm organic filter membrane, and detect the amount of acrylamide generated in the liquid phase.

[0038] Detection of acrylamide: high performance liquid chromatography, the mobile phase is methanol: water = 5:95, Pntulips QS-C18Plus chromatographic column (4.6mm×250mm), column temperature is 40°C; the flow rate is 1mL / min.

[0039] Product tolerance determination: The initial NHAM and mutants were immersed and incubated in a 500 g / L acrylamide solution at 28°C for 30 min and the enzyme activity was measured. The enzyme activity of the enzyme without immersion treatment was defined as 100%, and the tolerance results in the 500 g / L acrylamide solution were obtained.

[0040] In the present invention, the amino acid sequence of the α subunit of nitrile hydratase NHAM is shown in SEQ ID NO.1, the amino acid sequence of the β subunit of nitrile hydratase NHAM is shown in SEQ ID NO.2, the amino acid sequence of the regulatory protein of nitrile hydratase NHAM is shown in SEQ ID NO.3, the gene encoding the β subunit mutation is shown in SEQ ID NO.5, the gene encoding the α subunit mutation is shown in SEQ ID NO.4, the regulatory protein gene is shown in SEQ ID NO.6, and the connection sequence between the β subunit and the regulatory protein p14k is shown in SEQ ID NO.7, which is specifically as follows:

[0041] SEQ ID NO.1:

[0042] MTGSHGRDGDHHGHHHDRDHDNHLDPMTARVMALETILTEKGMVDPDALDAIIDTYETKVGPRNGASVVAKAWSDPDYADWLARDATAAIASLGFTGRQGEHMQAVFNTP ERHNLVVCTLCSCYPWSVLGLPPVWYKSPPYRSRAVSDPRGVLREFGVALPDGVSVRVWDSTAELRYLVVPERPAGTEGLSEAALAALVTRKSMIGTERDLSPHAAPETAA

[0043] SEQ ID NO.2:

[0044] MNGPHDLGGRHGFGPIAPKADEPLFHAPWERRALALTLAAGAMGHWSIDESRAAREDRHPADYYGSSYYEIWTKGLETLLLRHGLISHRELRAGRPLDLTVPPNRILKADAVAPALAKGSPANRDPEGSTPVFAPGDRVRTLNLQPRHHIRLPAYAREKAGTIETVQGFHVFADASAKGDDHVAHWLYTVVFDAFTLWGGDASPNDTVSIDAWEPYLAHA

[0045] SEQ ID NO.3:

[0046] MSKIKPGSPILRTPETGIAASPGLPRDAAGEPVFFAPWQAKAFAMTVALNERGILAWTDWAAALGRACASLPAAGPSPEATADAYFTAWLVALEEILTARALVSANAVDAAQAVWHRAAEATPHGTPIRFEAGLPNPHD

[0047] SEQ ID NO.4:

[0048] ATGACGGGATCGCACGGCAGGGACGGTGATCACCACGGCCATCACCACGACCGTGATCACGACAACCATCTCGACCCGATGACCGCGCGGGTCATGGCGCTGGAGACGATCCTCACCGAAAAGGGCATGGTCGACCCGGACGCCCTCGACGCCATCATCGACACCTACGAGACCAAGGTCGGGCCGCGCAACGGCGCCAGCGTCGTCGCCAAGGCCTGGAGCGACCCGGACTACGCCGACTGGCTGGCGCGCGACGCAACCGCCGCCATTGCCTCGCTTGGCTTCACCGGCCGCCAGGGCGAGCACATGCAGGCGGTGTTCAACACCCCGGAGCGCCACAACCTCGTCGTCTGCACCCTGTGCTCCTGCTATCCGTGGTCAGTGCTCGGCCTGCCGCCGGTCTGGTACAAGTCGCCGCCCTATCGCTCGCGCGCCGTCTCCGATCCGCGCGGCGTCCTGCGCGAATTCGGCGTCGCGCTGCCGGACGGCGTCTCGGTGCGAGTCTGGGACTCCACCGCCGAGCTGCGCTACCTCGTCGTGCCCGAGCGCCCGGCGGGTACCGAGGGACTGTCCGAGGCGGCGCTGGCGGCGCTCGTCACCCGCAAGTCCATGATCGGTACCGAGCGTGACCTGAGCCCGCATGCAGCACCGGAGACAGCAGCATGA

[0049] SEQ ID NO.5:

[0050] ATGAACGGCCCCCACGATCTCGGCGGTCGGCACGGCTTCGGGCCGATCGCGCCGAAGGCAGACGAGCCGCTGTTCCATGCGCCCTGGGAGCGCCGCGCCCTCGCCCTGACGCTCGCCGCCGGTGCGATGGGCCATTGGTCGATCGACGAAAGCCGCGCCGCCCGTGAGGATCGCCACCCGGCCGACTATTACGGTTCGTCCTATTACGAGATCTGGACCAAGGGCCTTGAGACGCTGCTCCTGCGCCACGGCCTCATCAGCCATCGCGAATTGCGCGCCGGGCGGCCCCTCGACCTGACCGTGCCGCCGAACCGCATCCTGAAGGCCGATGCCGTCGCGCCGGCCCTTGCCAAGGGCAGTCCGGCCAACCGCGATCCCGAAGGCAGCACGCCCGTTTTCGCGCCGGGCGACAGGGTCCGCACGCTGAACCTGCAGCCGCGCCATCACATCCGCCTGCCCGCCTATGCCCGCGAGAAGGCCGGCACCATCGAAACCGTTCAGGGTTTCCATGTCTTCGCGGATGCCAGCGCCAAGGGCGACGACCATGTCGCGCACTGGCTCTACACGGTGGTCTTCGACGCATTCACGCTGTGGGGCGGCGACGCTTCGCCCAACGACACCGTCTCCATCGATGCCTGGGAGCCCTATCTTGCGCACGCCTGA

[0051] SEQ ID NO.6:

[0052] ATGTCTAAAATAAAACCTGGGAGCCCTATCTTGCGCACGCCTGAGACCGGCATCGCCGCATCGCCCGGCCTGCCACGCGATGCGGCGGGTGAACCCGTCTTCTTCGCGCCCTGGCAGGCCAAGGCCTTCGCCATGACCGTCGCGCTGAACGAGCGCGGCATCCTTGCCTGGACCGACTGGGCTGCCGCGCTCGGCCGCGCCTGCGCCAGC CTGCCCGCCGCCGGCCCCTCGCCCGAAGCAACAGCGGATGCCTATTTCACCGCATGGCTCGTCGCGCTCGAAGAAATCCTCACGGCACGGGCGCTGGTAAGCGCCAATGCCGTCGACGCGGCGCAGGCCGTCTGGCACCGCGCCGCCGAGGCCACGCCCCACGGCACGCCGATCCGCTTCGAGGCCGGCCTGCCGAACCCACACGACTGA

[0053] SEQ ID NO.7:

[0054] AAGGAGATATACAT.

[0055] Example 1 Construction of initial NHAM and mutant recombinant bacteria

[0056] The nitrile hydratase NHAM from Aurantimonas manganoxydans SI859A was subjected to iterative saturation mutagenesis, and mutations were designed at positions 41, 51 and 55 of the β subunit and at position 98 of the α subunit.

[0057] Beijing Qingke Xinye Biotechnology Co., Ltd. was commissioned to provide codon optimization and gene synthesis services, and the genes of the initial type and mutant of nitrile hydratase NHAM from Aurantimonas manganoxydans SI859A were cloned into the pET-28a(+) plasmid, and the target gene was placed between the restriction sites BamH I and Hind III. The pET-28a(+)-NHAM recombinant plasmid was obtained.

[0058] Using pET-28a(+)-NHAM as a template, PCR was performed under the conditions shown in Table 1. The PCR product was transformed into E. coli BL21 (DE3) and sequenced by Beijing Qingke Xinye Biotechnology Co., Ltd. The plasmid with the correct sequencing result was expressed to obtain a recombinant strain.

[0059] Table 1 PCR amplification system

[0060]

[0061]

[0062] PCR amplification conditions:

[0063] 1) Pre-denaturation: 98℃ for 5min;

[0064] 2) Denaturation: 98°C for 10 s; annealing: 60°C for 15 s; extension: 72°C for 1 min for 30 s; 35 cycles in total;

[0065] 3) Extension: 72°C for 10 min;

[0066] 4) Store at 4℃ for 2.0h.

[0067] After PCR amplification, the amplified product was detected by 1.0% agarose gel electrophoresis, and the target band was purified and recovered using a DNA recovery and purification kit.

[0068] The recovered PCR product was digested with DPNI to remove the template. The digestion system is shown in Table 2.

[0069] Table 2 Digestion system

[0070] Reagents Volume (μL) PCR amplification product / plasmid 8.5 DPNI 0.5 10×Buffer 1

[0071] Digestion conditions:

[0072] 1) 37°C: 1h;

[0073] 2) 75℃: 15min;

[0074] 3) Store at 4°C for 2.0h.

[0075] The digested products were transfected into E. coli BL21 competent cells.

[0076] Example 2 Microbial culture and enzyme activity determination

[0077] (1) Cultivation of microorganisms

[0078] LB liquid medium composition: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, dissolved in deionized water and fixed to volume, sterilized at 121℃ for 20min, and set aside. LB solid medium (plate culture dish): add 20g / L agar powder to LB liquid medium, sterilize at 121℃, cool down and introduce into the culture dish to make a plate.

[0079] Inoculate the engineered bacteria E.coli BL21 (DE3) containing the relevant gene into 5 mL LB liquid medium containing 50 μg / mL kanamycin, and shake culture at 37°C for 12 hours. Transfer to 500 mL of fresh LB liquid medium also containing 50 μg / mL Kan, shake culture at 37°C until OD600 reaches about 0.8, add IPTG to a concentration of 0.5 mM, add cobalt chloride to a final concentration of 0.4 mM, and induce culture at 18°C ​​for 16-18 hours. After the culture is completed, centrifuge the culture solution at 4000 rpm for 10 minutes, discard the supernatant, collect the bacterial cells, and store them in a -70°C ultra-low temperature refrigerator for later use.

[0080] (2) Determination of enzyme activity

[0081] The bacterial cells collected after the culture was completed were washed twice with 0.25M PB buffer (pH 7.5), and then resuspended in 0.25M PB buffer (pH 7.5) with 1 times the volume of the fermentation liquid, and the cell suspension was used for subsequent measurements.

[0082] Definition of enzyme activity: The International Conference on Enzymology in 1961 stipulated that one unit of enzyme activity refers to the amount of enzyme that can convert 1 micromole of substrate within 1 minute under specific conditions, or the amount of enzyme that converts 1 micromole of related groups in the substrate.

[0083] Nitrile hydratase activity assay system: the total system is 500 μL, the reaction medium is 0.25 M phosphate buffer at pH 7.5, the substrate is 425 μL 50 g / L acrylonitrile, 50 μL of bacterial solution with OD600=4 is added, and after reacting at 28°C for 5 minutes, 25 μL 4 mol / L HCl is added to terminate the reaction.

[0084] The reaction solution was centrifuged at 12000 rpm for 3 min, the supernatant was taken out and diluted 10 times, and the amount of acrylamide produced was determined by liquid chromatography.

[0085] Example 3 Tolerance determination of NHAM initial type and mutants

[0086] Product tolerance of enzyme: The collected cells were resuspended in 500g / L acrylamide solution, immersed in a 28°C water bath for 1h, and then the residual enzyme activity was determined using an enzyme activity detection system. The enzyme activity of the untreated enzyme was defined as 100%. After the NHAM initial type was immersed in 500g / L acrylamide solution for 30min, no residual enzyme activity was detected. Residual enzyme activity could be detected for multiple point mutants. The mutant β-S51E / R98I had the highest residual enzyme activity of 54.07%, and the initial enzyme activity was significantly improved compared to the wild type. The four-point combination mutant β-R55C / β-S51W / β-G41D / α-R98I showed the highest initial enzyme activity, which was 6 times that of the wild type, and the residual enzyme activity after acrylamide treatment was 42.62%. The initial enzyme activity and acrylamide tolerance of all mutants are shown in the figure below. Figure 1 As shown, it can be seen that all mutants have good tolerance to the substrate acrylamide.

[0087] Example 4 Determination of substrate spectrum of NHAM initial type and mutants

[0088] The reaction system is 0.5 mL, 0.25 M phosphate buffer (pH 7.5) containing 100 mM substrate, add an appropriate amount of enzyme solution to start the reaction, 28 ° C, 700 rpm metal bath reaction for 5 minutes, immediately add 0.5 mL of pure acetonitrile to terminate the reaction, the reaction solution is centrifuged at 12000 rpm for 3 minutes, and then filtered through a 0.22 μm organic filter membrane. The amount of the product is detected by high performance liquid chromatography, and the results are as follows Figure 2 As shown, the catalytic activities of the 13 combined mutants on isobutyronitrile, adiponitrile, 2-butenenitrile, 3-cyanopyridine, 4-cyanopyridine and benzonitrile were significantly improved. The catalytic activities of each mutant on different substrates were measured, and the results are shown in Table 3.

[0089] Table 3 Types and catalytic activities of various mutants

[0090]

[0091] Among them, the liquid chromatography detection conditions are: chromatographic column Pntulips QS-C18 Plus chromatographic column (4.6mm×250mm), column temperature 40°C; flow rate 1mL / min; different product detection conditions are as follows:

[0092] Table 4 Liquid phase detection conditions for different products.

[0093]

[0094] Example 5NHAM mutant catalyzes acrylonitrile to produce acrylamide

[0095] Take the fermentation broth of the genetically engineered bacteria constructed in Example 2, centrifuge at 4000rpm for 10min to collect the cells, add 100000U of enzyme, and resuspend in phosphate buffer (pH 7.5), a total of 194.5g, hydration reaction at 20°C, add 40g / L acrylonitrile to each batch, and put into the next batch after the substrate is completely consumed. Then use gas chromatography to detect the content of acrylonitrile, acrylamide and acrylic acid in the reaction system. The substrate conversion rate is greater than 99%, the acrylamide concentration reaches 482.5g / L, and no acrylic acid is generated in the reaction system.

[0096] Example 6 NHAM mutant catalyzes isobutyronitrile to produce isobutyramide

[0097] Take the fermentation broth of the genetically engineered bacteria constructed in Example 2, centrifuge at 4000rpm for 10min to collect the cells, add 100000U of enzyme, and resuspend in phosphate buffer (pH 7.5), a total of 194.5g, hydration reaction at 25°C, and add 100g / L isobutyronitrile in batches. After 8h of reaction, the content of isobutyronitrile and isobutyramide in the reaction system was detected by liquid chromatography. The substrate residue was 0.4g / L, and the substrate conversion rate was 99.6%.

[0098] Example 7NHAM mutant catalyzes adiponitrile to produce adipic acid diamide

[0099] The fermentation broth of the genetically engineered bacteria constructed in Example 2 was taken, centrifuged at 4000rpm for 10min to collect the cells, and 100000U of enzyme was added to the cells, and phosphate buffer (pH 7.5) was resuspended to a total of 200g, and hydration reaction was carried out at 25°C, and 50g / L of adiponitrile was added. After reacting for 12h, the contents of adiponitrile and adipic acid diamide in the reaction system were detected by liquid chromatography. No substrate residue was detected, and the substrate conversion rate was greater than 99.9%.

[0100] Example 8 NHAM mutant catalyzes 2-butenenitrile to form crotonamide

[0101] Take the fermentation broth of the genetically engineered bacteria constructed in Example 2, centrifuge at 4000rpm for 10min to collect the cells, add 100000U of enzyme, and resuspend in phosphate buffer (pH 7.5), a total of 195.5g, hydration reaction at 25°C, add 50g / L 2-butenenitrile. After 24h of reaction, the content of 2-butenenitrile and crotonamide in the reaction system was detected by liquid chromatography. No substrate residue was detected, and the substrate conversion rate was greater than 99.9%.

[0102] Example 9 NHAM mutant catalyzes 3-cyanopyridine to produce nicotinamide

[0103] Take the fermentation broth of the genetically engineered bacteria constructed in Example 2, centrifuge at 4000rpm for 10min to collect the cells, add 100000U of enzyme, and resuspend in phosphate buffer (pH 7.5), a total of 197.5g, hydration reaction at 20°C, add 70g / L 3-cyanopyridine to each batch, and put into the next batch after the substrate is completely consumed. Then use liquid chromatography to detect the content of 3-cyanopyridine and nicotinamide in the reaction system. The concentration of nicotinamide reaches 402.1g / L, and the substrate conversion rate is greater than 99.9%.

[0104] Example 10NHAM mutant catalyzes 4-cyanopyridine to produce isonicotine

[0105] Take the fermentation broth of the genetically engineered bacteria constructed in Example 2, centrifuge at 4000rpm for 10min to collect the cells, add 100000U of enzyme, and resuspend in phosphate buffer (pH 7.5), a total of 196.5g, hydration reaction at 20°C, add 70g / L 4-cyanopyridine to each batch, and put into the next batch after the substrate is completely consumed. Then use liquid chromatography to detect the content of 4-cyanopyridine and isonicotine in the reaction system. The isonicotine concentration reaches 415.3g / L, and the substrate conversion rate is greater than 99.9%.

[0106] Example 11NHAM mutant catalyzes the formation of benzyl group into benzamide

[0107] The fermentation broth of the genetically engineered bacteria constructed in Example 2 was taken, centrifuged at 4000rpm for 10min to collect the cells, and 100000U of enzyme was added to the cells, and phosphate buffer (pH 7.5) was resuspended to a total of 195.5g, and hydration reaction was carried out at 30°C, and 100g / L benzyl was added. After reacting for 6h, the contents of benzyl and benzamide in the reaction system were detected by liquid chromatography. The substrate residue was 0.7g / L, and the substrate conversion rate was 98.6%.

[0108] Example 12NHAM mutant catalyzes 2,6-difluorobenzonitrile to produce 2,6-difluorobenzamide

[0109] Take the fermentation broth of the genetically engineered bacteria constructed in Example 2, centrifuge at 4000rpm for 10min to collect the cells, add 100000U of enzyme, and resuspend in phosphate buffer (pH 7.5), a total of 200.5g, hydration reaction at 30°C, and add 50g / L 2,6-difluorobenzonitrile. After 12h of reaction, the content of 2,6-difluorobenzonitrile and 2,6-difluorobenzamide in the reaction system was detected by liquid chromatography. No substrate residue was detected, and the substrate conversion rate was greater than 99.9%.

[0110] Comparative Example 1

[0111] According to the same method as in Example 2, genetically engineered bacteria E. coli BL21 (DE3) -pET-28a (+) -NHAM-β-G41D, E. coli BL21 (DE3) -pET-28a (+) -NHAM-β-S51E, E. coli BL21 (DE3) -pET-28a (+) -NHAM-β-R55C, and E. coli BL21 (DE3) -pET-28a (+) -NHAM-α-R98I with single-point mutations in nitrile hydratase were constructed, and their enzyme activities were measured. The results are shown in the following table.

[0112] Table 5

[0113]

Claims

1. A nitrile hydratase mutant, comprising an α subunit, a β subunit and a regulatory protein, characterized in that: The amino acid sequence of the regulatory protein is shown in SEQ ID NO.3; the α subunit and β subunit of the nitrile hydratase mutant are any one of the following (1) to (13): (1) mutating the 98th arginine of the α subunit of the nitrile hydratase whose amino acid sequence is shown in SEQ ID NO.1 to isoleucine, and mutating the 41st glycine of the β subunit of the nitrile hydratase whose amino acid sequence is shown in SEQ ID NO.2 to aspartic acid; (2) mutating the 98th arginine of the α subunit of the nitrile hydratase with an amino acid sequence as shown in SEQ ID NO.1 to valine, and mutating the 41st glycine of the β subunit of the nitrile hydratase with an amino acid sequence as shown in SEQ ID NO.2 to aspartic acid; (3) The serine at position 51 of the β subunit of the nitrile hydratase having the amino acid sequence as shown in SEQ ID NO.2 is mutated to glutamic acid, and the arginine at position 98 of the α subunit is mutated to valine; (4) mutating the serine at position 51 of the β subunit of the nitrile hydratase having the amino acid sequence as shown in SEQ ID NO.2 to glutamic acid, and mutating the arginine at position 98 of the α subunit to isoleucine; (5) mutating the arginine at position 98 of the α subunit of the nitrile hydratase with an amino acid sequence as shown in SEQ ID NO.1 to valine, and mutating the arginine at position 55 of the β subunit of the nitrile hydratase with an amino acid sequence as shown in SEQ ID NO.2 to cysteine; (6) mutating the serine at position 51 of the β subunit of the nitrile hydratase having an amino acid sequence as shown in SEQ ID NO.2 to tryptophan, and mutating the arginine at position 55 to cysteine; (7) mutating the glycine at position 41 of the β subunit of the nitrile hydratase having the amino acid sequence as shown in SEQ ID NO.2 to aspartic acid, the serine at position 51 to tryptophan, and the arginine at position 55 to cysteine; (8) mutating the glycine at position 41 of the β subunit of the nitrile hydratase having an amino acid sequence as shown in SEQ ID NO.2 to serine, the serine at position 51 to tryptophan, and the arginine at position 55 to cysteine; (9) mutating the arginine at position 98 of the α subunit of the nitrile hydratase with an amino acid sequence as shown in SEQ ID NO.1 to isoleucine, and mutating the glycine at position 41 of the β subunit of the nitrile hydratase with an amino acid sequence as shown in SEQ ID NO.2 to aspartic acid, and mutating the serine at position 51 to valine; (10) mutating the arginine at position 98 of the α subunit of the nitrile hydratase with an amino acid sequence as shown in SEQ ID NO.1 to valine, and mutating the arginine at position 55 of the β subunit of the nitrile hydratase with an amino acid sequence as shown in SEQ ID NO.2 to cysteine, and mutating the serine at position 51 to asparagine; (11) mutating the arginine at position 98 of the α subunit of the nitrile hydratase with an amino acid sequence as shown in SEQ ID NO.1 to methionine, and mutating the glycine at position 41 of the β subunit of the nitrile hydratase with an amino acid sequence as shown in SEQ ID NO.2 to aspartic acid, the serine at position 51 to tryptophan, and the arginine at position 55 to cysteine; (12) mutating the arginine at position 98 of the α subunit of the nitrile hydratase with the amino acid sequence as shown in SEQ ID NO.1 to isoleucine, and mutating the glycine at position 41 of the β subunit of the nitrile hydratase with the amino acid sequence as shown in SEQ ID NO.2 to aspartic acid, the serine at position 51 to tryptophan, and the arginine at position 55 to cysteine; (13) The arginine at position 98 of the α subunit of the nitrile hydratase whose amino acid sequence is shown in SEQ ID NO.1 is mutated to leucine, and the glycine at position 41 of the β subunit of the nitrile hydratase whose amino acid sequence is shown in SEQ ID NO.2 is mutated to aspartic acid, the serine at position 51 is mutated to tryptophan, and the arginine at position 55 is mutated to cysteine.

2. A gene encoding the nitrile hydratase mutant according to claim 1.

3. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the gene according to claim 2.

4. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria comprises the gene as claimed in claim 2, or comprises the recombinant expression vector as claimed in claim 3.

5. An enzyme preparation, characterized in that: The invention comprises the nitrile hydratase mutant according to claim 1.

6. Use of the nitrile hydratase mutant according to claim 1, or the genetically engineered bacteria according to claim 4, or the enzyme preparation according to claim 5 in catalyzing the production of amides from aliphatic or aromatic nitrile compounds, characterized in that: Aliphatic nitrile compounds include acrylonitrile, isobutyronitrile, adiponitrile, and 2-butenenitrile; aromatic nitrile compounds include 3-cyanopyridine, 4-cyanopyridine, benzonitrile, and 2,6-difluorobenzonitrile.

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