Nitrile hydratase with improved activity and thermal stability and application thereof
By mutation of specific sites of nitrile hydratase, its activity and thermal stability are improved, the existing problems of low activity and poor stability of nitrile hydratase are solved, and more efficient synthesis of amide compounds is achieved.
Patent Information
- Application Number
- CN202411616294.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-13
- Publication Date
- 2025-02-07
AI Technical Summary
The existing nitrile hydratase has low activity and poor stability, which limits the industrialization level of efficient synthesis and production of amide compounds.
The catalytic activity and thermal stability are enhanced by mutations at specific sites such as G86F, K57F, E183F and T149I, G164T, G208P of the nitrile hydratase.
The thermal stability of nitrile hydratase was significantly improved, and the half-life at 65°C was increased from 21 minutes to 210 minutes, while improving catalytic activity.
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Abstract
Description
Technical Field
[0001] The invention relates to a nitrile hydratase with improved activity and thermal stability and application thereof, belonging to the technical field of bioengineering. Background Art
[0002] Nitrile hydratase (NHase, EC 4.2.1.84) is a metalloenzyme that can hydrate nitrile substrates to convert them into corresponding amide products. It is mainly used in the industrial production of acrylamide and nicotinamide. Amide compounds have been widely used in food, medicine, cosmetics, feed and fine chemical industries. However, the hydration reaction of nitrile hydratase is an exothermic reaction. In industrial production, it is usually necessary to maintain a low temperature (25°C) to maintain strong catalytic performance, which increases production costs. In addition, natural nitrile hydratase has low activity, poor stability and poor tolerance to organic substrates / products, which restricts the efficient synthesis of high-value amide compounds and the industrialization level of amide product production. The heat-resistant nitrile hydratase (PtNHase) from Pseudonocardia thermophila JCM3095 can reach nearly 90% residual enzyme activity after treatment at 60°C for 5h, which has excellent thermal stability, but the catalytic enzyme activity of the wild type is low. Therefore, it is of great significance to obtain nitrile hydratase with both high activity and good thermal stability. Many studies have been reported to improve the performance of nitrile hydratase by molecular modification of nitrile hydratase to solve the problem of poor stability of nitrile hydratase.
[0003] For example, protein fragment swapping of nitrile hydratase improves its thermal stability (Cui Y, Cui W, Liu Z, et al. Improvement of stability of nitrile hydratase via protein fragment swapping [J]. Biochemical & Biophysical Research Communications, 2014, 450 (1): 401-408.); the C-terminal domain of nitrile hydratase is swapped to obtain a chimeric nitrile hydratase, which greatly improves its thermal stability (Sun W, Zhu L, Chen X, et al. The Stability Enhancement of Nitrile Hydratase from Bordetella petrii by Swapping the C-terminal Domain of β subunit [J]. Applied Biochemistry and Biotechnology, 2016, 178 (8): 1481-1487.); using FireProt server prediction and MD simulation, site-directed mutagenesis of nitrile hydratase was performed to obtain ten-point mutants, whose activity and thermal stability were improved (Cheng Z, Lan Y, Guo J, et al. Computational Design of Nitrile Hydratasefrom Pseudonocardia thermophila JCM3095 for Improved Thermostability[J]. Molecules, 2020(20).). The reported strategies for improving the thermal stability of nitrile hydratase mainly include salt bridge introduction, thermal stability fragment exchange, domain exchange and subunit fusion.
[0004] Enzyme engineering is an effective means to improve the industrial properties of enzymes (activity, stability, tolerance, selectivity, etc.), mainly including three strategies: directed evolution, rational design, and semi-rational transformation. Rational design is an important method to improve the performance of biomacromolecules such as protein stability, tolerance, and enzyme catalytic selectivity. After in-depth analysis of detailed information such as the three-dimensional structure, function, and mechanism of the protein, mutation sites are designed. However, it is often limited by limited computing power and the accuracy of the computational model, making it difficult to ensure the mutation calculation space and design accuracy. Summary of the invention
[0005] The present invention provides a nitrile hydratase mutant with improved catalytic activity and / or thermal stability, which is based on the parent and has mutations (a) and / or (b):
[0006] (a) mutating one or more of positions 57, 86, and 183 of the α-subunit of nitrile hydratase;
[0007] (b) mutating one or more of positions 149, 164, and 208 of the β-subunit of nitrile hydratase.
[0008] In one embodiment, the parent has an α subunit as shown in SEQ ID NO.1 and a β subunit as shown in SEQ ID NO.3.
[0009] In one embodiment, the nitrile hydratase mutant is a mutant of the parent enzyme, wherein the glycine at position 86 of the sequence shown in SEQ ID NO.1 is mutated to phenylalanine, and is named αG86F.
[0010] In one embodiment, the nitrile hydratase mutant is based on the parent, with the 57th lysine in the sequence shown in SEQ ID NO.1 mutated to phenylalanine, and the 183rd glutamic acid mutated to phenylalanine, and is named αK57F-αE183F.
[0011] In one embodiment, the nitrile hydratase mutant is based on the parent, with the glycine at position 86 mutated to phenylalanine, the lysine at position 57 mutated to phenylalanine, and the glutamate at position 183 mutated to phenylalanine in the sequence shown in SEQ ID NO.1, and is named αG86F-αK57F-αE183F.
[0012] In one embodiment, the nitrile hydratase mutant is a mutant of the parent enzyme, wherein the threonine at position 149 of the sequence shown in SEQ ID NO. 3 is mutated to isoleucine, and is named βT149I.
[0013] In one embodiment, the nitrile hydratase mutant is a mutant of the parent enzyme, wherein the glycine at position 164 of the sequence shown in SEQ ID NO. 3 is mutated to threonine, and is named βG164T.
[0014] In one embodiment, the nitrile hydratase mutant is based on the parent, with the threonine at position 149 of the sequence shown in SEQ ID NO.3 mutated to isoleucine, and the glycine at position 208 mutated to proline, and is named βT149I-βG208P.
[0015] In one embodiment, the nitrile hydratase mutant is based on the parent, with the threonine at position 149 mutated to isoleucine, the glycine at position 164 mutated to threonine, and the glycine at position 208 mutated to proline in the sequence shown in SEQ ID NO.3, and is named βT149I-βG164T-βG208P.
[0016] The present invention also provides a gene encoding the nitrile hydratase mutant.
[0017] The present invention also provides a recombinant microbial cell expressing the nitrile hydratase mutant.
[0018] The invention also provides a recombinant Escherichia coli expressing the nitrile hydratase mutant.
[0019] In one embodiment, the recombinant Escherichia coli uses BL21 (DE3) as a host and pET24a as a vector to express the nitrile hydratase mutant.
[0020] The present invention also provides a method for improving the catalytic activity and / or thermal stability of nitrile hydratase, which comprises performing one or more mutations of G86F, K57F, and E183F on the α subunit of nitrile hydratase, and / or performing one or more mutations of T149I, G164T, and G208P on the β subunit of nitrile hydratase.
[0021] In one embodiment, the α subunit of the nitrile hydratase has an amino acid sequence shown in SEQ ID NO.1.
[0022] In one embodiment, the β subunit of the nitrile hydratase has an amino acid sequence shown in SEQ ID NO.3.
[0023] The present invention also provides a method for preparing the nitrile hydratase mutant, wherein the recombinant Escherichia coli is cultured in a culture medium for a period of time, and IPTG and Co 2+ Induce enzyme expression.
[0024] The present invention also provides the use of the nitrile hydratase mutant, or the gene, or the recombinant microbial cell, or the recombinant Escherichia coli in preparing amide compounds.
[0025] In one embodiment, the application includes but is not limited to the application in the preparation of acrylamide, nicotinamide or products containing acrylamide, or products containing nicotinamide.
[0026] Beneficial effects:
[0027] The present invention performs computational design on the dimer interface, tetramer interface and amino acid co-evolution pairs within the subunit of nitrile hydratase, constructs mutations, and performs screening, and finally obtains nitrile hydratase mutants with improved activity and thermal stability. The thermal stability of the mutant constructed by the present invention is significantly improved, and the half-life at 65° C. is increased from 21 minutes of the wild type to 210 minutes. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 Whole-cell activity thermostability assay for forward mutations at the dimer interface.
[0029] Figure 2 Whole-cell activity thermostability assay for tetramer interface forward mutations.
[0030] Figure 3 Whole-cell activity thermostability assay for coevolution of forward mutants.
[0031] Figure 4 The combined mutant pure enzyme assay and thermal stability assay. a: Pure enzyme specific enzyme activity thermal stability assay; b: Combined mutant pure enzyme half-life assay. DETAILED DESCRIPTION
[0032] 1. Gene amplification and fragment purification
[0033] (1) PCR reaction system (50 μL): 25 μL PrimeSTAR Max Premix (2×) (Takara), 2 μL each of upstream and downstream primers (10 μM), 0.5 μL DNA template, and 20.5 μL ddH2O.
[0034] (2) PCR reaction procedure: pre-denaturation at 98°C for 1 min; 30 cycles of 98°C for 30 s, 55°C for 15 s, and 72°C for 1 min; and full extension at 72°C for 5 min.
[0035] (3) DpnI digestion to remove template DNA: 1 μL restriction endonuclease DpnI (Takara) and 5 μL 10× Buffer were added to the PCR reaction product and placed in a 37°C metal bath for 1 h. The digested reaction solution was purified using a Gel Extraction Kit (Kangwei Century) to obtain a high-purity PCR amplification product.
[0036] 2. Plasmid construction based on seamless cloning
[0037] (1) Fragment assembly. Prepare a DNA seamless ligation reaction system (10 μL): 5 μL 2X MultiF Seamless Assembly Mix (ABclonal), 2.5 μL each of DNA gene fragment and vector fragment. React in a 55°C metal bath for 30 min.
[0038] (2) Transformation. The seamless cloning reaction solution was transformed into E. coli BL21 ER2566 competent cells. After 1 h of recovery culture, it was spread on a solid LB plate containing 50 μg mL-1 kanamycin and cultured overnight at 37°C to obtain transformants. Single colonies were picked for subsequent verification.
[0039] 3. Culture medium
[0040] LB medium (L -1 ): 10g tryptone, 5g yeast extract, 10g NaCl, pH 7.0, add 15g agar powder when preparing solid culture medium.
[0041] The antibiotic concentration used during culture was kanamycin (50 μg / mL).
[0042] 4. HPLC determination of nitrile hydratase activity
[0043] The chromatographic column was Diamonsil C18(2) 5μm 250x 4.6mm (Dima Technology, China), and the mobile phase was a mixture of acetonitrile and water [the volume ratio was V(water):V(acetonitrile)=2:1]. The detection wavelength was 215nm, the column temperature was 40℃, and the mobile phase flow rate was 0.6mL·min -1 , injection volume 10μL.
[0044] Unit enzyme activity (U) is defined as the amount of enzyme required to catalyze acrylonitrile to produce 1 μmol acrylamide per minute at 25°C. -1 ) is defined as the enzyme activity per mg of nitrile hydratase.
[0045] 5. Calculation of free energy change of amino acid mutation
[0046] The Cartesian_ddG module of Rosetta software was used to calculate the folding free energy change of protein residue mutations. The InterfaceAnalyzer module of Rosetta software was used to analyze the protein subunit interface and extract the amino acid residue information located at the subunit interface. The Flex_ddG module of Rosetta software was used to calculate the affinity change between protein subunits after residue mutation.
[0047] Example 1: Mutation design of PtNHase subunit interface
[0048] According to the crystal structure of PtNHase (PDB ID: 1IRE), it is a heterotetrameric protein, and the α subunit (shown in SEQ ID NO.1) and the β subunit (shown in SEQ ID NO.3) form a heterodimer, which is further combined with another α / β dimer unit to form a heterotetramer. Therefore, PtNHase has two subunit interfaces, dimer and tetramer. The subunit interface was analyzed using the InterfaceAnalyzer module of Rosetta software to determine the amino acid residues that form the subunit interface (Table 1). Saturation mutations were designed for the amino acids at the subunit interface (excluding key functional residues such as active centers), and the changes in affinity between subunits after mutation were calculated. For the dimer interface, the α / β dimer was used as a model to calculate the changes in affinity between the α subunit and the β subunit before and after the mutation; for the tetramer interface, the α2 / β2 tetramer was used as a model to calculate the changes in affinity between the two α / β dimer units before and after the mutation. Potential positive mutations were screened using a threshold of ΔΔG ≤ -1.0. The results are shown in Table 2.
[0049] Table 1 PtNHase interface amino acids
[0050]
[0051]
[0052] Table 2 Interface mutations that improve inter-subunit affinity
[0053]
[0054]
[0055] Example 2: Construction and screening of PtNHase dimer and tetramer interface mutations
[0056] (1) Construction of mutants:
[0057] Using pET24a-PtNHase plasmid as a template (published in Li, M., Ma, D., Qiao, J. et al. Development of high-performance nitrile hydratase whole-cell catalyst by automated structure-and sequence-based design and mechanism insights. Syst Microbiol and Biomanuf 4, 882–894 (2024). The nucleotide sequence encoding the α subunit is shown in SEQ ID NO.2, and the nucleotide sequence encoding the β subunit is shown in SEQ ID NO.4), single-point mutations of interface residues were constructed using the primers listed in Table 3, and the cells were transformed into BL21 (DE3) strains for induced expression.
[0058] (2) The recombinant bacteria obtained by the construction are cultured under the following conditions to prepare bacterial cells:
[0059] The strain was inoculated into a test tube containing 3 mL of LB medium (containing kanamycin) and cultured at 37°C and 200 rpm for 12 h. A 1% inoculum was transferred to a test tube containing 5 mL of LB medium (containing kanamycin) and cultured at 37°C and 200 rpm until OD 600 ≈0.6, add IPTG with a final concentration of 0.5mM and cobalt chloride hexahydrate (CoCl2·6H2O) solution of 0.4mM, cool to 25℃, and continue to culture for 16h to fully induce the expression of nitrile hydratase.
[0060] (3) Using whole cells for activity and thermal stability screening:
[0061] The induced expression bacterial solution was centrifuged at 8000 rpm for 5 min, the supernatant was discarded, and the suspension was resuspended in 10 mM phosphate buffer (KPB). The OD was adjusted by spectrophotometer. 600 to 0.5 for activity and thermal stability testing.
[0062] Activity detection: Take 10μL of cell suspension into a 1.5mL EP tube, add 490μL of 100mM acrylonitrile solution and incubate in a 25℃ metal bath for 5min, add 500μL of 0.1M H3PO4 solution to terminate. Then filter through a 0.22μm organic microporous filter membrane. Use high performance liquid chromatography to detect the amount of acrylamide generated and calculate the whole cell catalytic activity.
[0063] Thermal stability test: The whole cell suspension was placed in a 65°C metal bath and incubated for 1 hour. The activity was measured according to the above-mentioned activity detection method. The ratio of the obtained activity to the initial activity was the residual activity.
[0064] like Figure 1 and Figure 2 Among them, the mutants with improved activity include αT18Y, αS26W, αG32D, αT35V, αD40C, αG90S, αP123Y, αE128S, αS133A, αE138K, αI157K, αT2M, αK8F, αE16G, αR137W, βA43Q, βP102D, βP102L, βP107I, βQ111D, βN2M, βN2Q, βV4Y, βG12Q, βG12C, βN59T, βP60M, βT149I, βT149H, βR160W, βG164M, βG164T, βG208P, βA230V, βA230R, and mutants with improved thermal stability include αG32D, βT149I, βG164T, βG164M, βG208P.
[0065] (4) Construction of double mutants
[0066] The mutants with improved thermal stability are freely combined in pairs, and mutations are introduced into the plasmid containing the single mutants according to the method of step (1) to obtain double mutants. The thermal stability and catalytic activity tests are performed according to the method of step (3) to screen and obtain the double point mutation βT149I-βG208P with excellent thermal stability and activity.
[0067] (5) Construction of triple mutants
[0068] Based on the double mutant constructed in step (4), the remaining mutations αG32D, βG164T, and βG164M were constructed into the double mutation in the form of single points or combinations, and the activity and thermal stability were tested according to the above method. The triple-point mutation M1 (βT149I-βG164T-βG208P) with the highest thermal stability and activity was obtained.
[0069] The constructed dimer and tetramer interface mutation primers are shown in Tables 3 and 4.
[0070] Table 3 Dimer interface mutation primers
[0071]
[0072]
[0073] Table 4 Tetramer interface mutation primers
[0074]
[0075]
[0076]
[0077] Example 3: Computational design of PtNHase co-evolution sites
[0078] The online tool EVcouplings was used to analyze the co-evolution sites of the sequences of the α subunit and β subunit of PtNHase, and the residue sites with co-evolution coupling were determined according to the scoring ranking. Sites with clear interactions (such as hydrogen bonds, salt bridges, hydrophobic interactions) and opposite side chain directions were excluded, and the remaining sites were used as computational design sites. The candidate residue pairs for mutation design are shown in Table 5. The residue pairs were subjected to saturated combination virtual mutations, and the Cartesian_ddG of Rosetta software was used to calculate the change in folding free energy after mutation. The mutation combinations that reduced the folding free energy were screened with ddG≤-1.0REU as the threshold, and redundant mutations with similar ddG changes and similar mutated residues were removed to obtain potential positive mutations, as shown in Table 6.
[0079] Table 5 Candidate residue pairs calculated for co-evolution sites
[0080]
[0081]
[0082] Table 6 Potential forward mutation designs for co-evolution sites
[0083]
[0084] Example 4: Construction and screening of PtNHase co-evolution site mutations
[0085] Using pET24a-PtNHase plasmid as template, co-evolution mutations were constructed using the primers listed in Table 7 and transformed into BL21 (DE3) strains. Expression was induced and whole cells were prepared according to Example 3, and activity and thermostability screening were performed to obtain mutants with improved activity or thermostability, such as Figure 3The mutations that improve activity include αG86F, αK57F-αE183F, αK61L-αE183F, αK61R-αE183F, αK61R-αE183Q, αM93T-αP117I, αM93L-αP117I, αP117I, βV133L-βA175V, βT181F-βC189F, βT181K-βC189Q, βR197Q-βS213Y, and the mutations that improve thermal stability include αG86F, αK57F-αE183F, αM93A-αP117Y, αM94V-αM166I, βT181K-βC189Q. Mutations αM93A-αP117Y and αM94V-αM166I were excluded due to their low activity. The remaining mutations αG86F, αK57F-αE183F, and βT181K-βC189Q were freely combined, constructed, and screened, and finally a three-point mutant M2 (αG86F-αK57F-αE183F) was obtained.
[0086] The constructed co-evolution mutation primers are shown in Table 7.
[0087] Table 7 Primers for co-evolution and mutation
[0088]
[0089]
[0090] Example 5: Iterative evolution of key distal residues
[0091] Using the plasmid of mutant M1 as a template, the point mutations of M2 were sequentially constructed onto M1. The operation steps were similar to those in Example 2, and a six-point combination mutation M3 (αG86F-αK57F-αE183F-βT149I-βG164T-βG208P) was obtained.
[0092] The mutation information of iterative evolution is shown in Table 8.
[0093] Table 8 Mutation information of iterative evolution
[0094] Mutants Mutation site M1 βT149I-βG164T-βG208P M2 αG86F-αK57F-αE183F M3 αG86F-αK57F-αE183F-βT149I-βG164T-βG208P
[0095] Example 6: Determination of pure enzyme properties of mutants
[0096] The wild type and mutants M1, M2, and M3 constructed in Example 5 were induced to overexpress, and the fermentation broth was collected and purified by affinity chromatography to determine the properties of the pure enzyme. The specific steps are:
[0097] Protein induction expression and ultrasonic disruption: Inoculate bacteria into a test tube containing 3 mL LB medium (kanamycin) and culture at 37°C, 200 rpm for 12 h. Transfer 1 mL of seed solution into a test tube containing 50 mL LB medium (containing kanamycin) and culture at 37°C, 200 rpm until OD 600 ≈0.6, add IPTG to a final concentration of 0.5mM and 0.4mM Co 2+ , cool to 25℃, continue to culture for 12-16h, so that nitrile hydratase can be fully induced to express. The induced bacterial solution is placed on ice to cool, and centrifuged at 8000rpm for 10min in a low-temperature centrifuge (4℃), and the supernatant is poured out to collect the bacterial precipitate. Resuspend the bacterial precipitate with 20mL Binding Buffer (10mM KPB, pH7.4), and crush it with an ultrasonic crusher for 20-30min. The bacterial suspension is always placed on ice during the crushing process. After crushing, centrifuge at 8000rpm for 20min, and filter the supernatant through a 0.22μm organic microporous filter membrane.
[0098] Protein purification by affinity chromatography: Since the nitrile hydratase expressed by the plasmid pET24a-PtNHase carries a Strep-tag at the C-terminus of the β subunit, the nitrile hydratase was purified using an AKTA protein purifier and a GE HisTrap HP 5mL column. First, the column was rinsed with pure water to remove ethanol, and then 5 to 10 column volumes of Binding Buffer were rinsed at a flow rate of 5 mL / min to balance the column, and then the sample was loaded through the loading line, and then eluted with 10-20 column volumes of Washing Buffer, and the target protein sample corresponding to the UV absorption peak was collected.
[0099] The protein concentration was quantified using a Bradford protein concentration assay kit, and the purification quality of the target protein was detected by SDS-PAGE.
[0100] Protein activity and half-life determination: Activity detection: adjust the protein concentration to 0.05 mg / mL, take 10 μL of enzyme solution into a 1.5 mL EP tube, add 490 μL of 100 mM acrylonitrile solution and incubate in a 25°C metal bath for 5 min, add 500 μL of 0.1 M H3PO4 solution to terminate. Then filter through a 0.22 μm organic microporous filter membrane. Use high performance liquid chromatography to detect the amount of acrylamide generated and calculate the specific enzyme activity.
[0101] Half-life determination: The enzyme solution (protein concentration is 0.05 mg / mL) was incubated in a 65°C metal bath. Samples were taken every 15 minutes for the first 90 minutes and every 120 minutes thereafter. The activity was measured using the above-mentioned activity detection method. The activity ratio obtained over the initial activity was the residual activity.
[0102] The results showed that the specific enzyme activities of WT, M1, M2 and M3 were 1656.84±21.21, 2370.17±38.75, 2265.07±36.13 and 2515.97±14.11 U / mg, respectively. The specific enzyme activity of M3 was 1.5 times that of WT. The half-lives of pure enzymes of WT, M1, M2 and M3 at 65°C were further determined. The results showed that the half-lives of WT, M1, M2 and M3 were 21min, 43min, 76min and 210min, respectively. M3 had significantly improved thermal stability ( Figure 4 b, Table 9).
[0103] Table 9 Characterization of mutant enzymes
[0104] Mutants Specific enzyme activity (U / mg) Half-life (min) WT 1656.84±102.21 21 M1 2370.17±58.75 43 M2 2265.07±46.13 76 M3 2515.97±74.11 210
[0105] 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 nitrile hydratase mutant having improved catalytic activity and / or thermal stability, characterized in that: Based on the parent, there are mutations of (a) and / or (b): (a) mutating one or more of positions 57, 86, and 183 of the α-subunit of nitrile hydratase; (b) mutating one or more of positions 149, 164, and 208 of the β-subunit of nitrile hydratase.
2. The nitrile hydratase mutant according to claim 1, characterized in that The parent has an α subunit as shown in SEQ ID NO.1 and a β subunit as shown in SEQ ID NO.
3.
3. The nitrile hydratase mutant according to claim 1, characterized in that The glycine at position 86 of the sequence shown in SEQ ID NO.1 was mutated to phenylalanine, the lysine at position 57 was mutated to phenylalanine, and the glutamate at position 183 was mutated to phenylalanine; the threonine at position 149 of the sequence shown in SEQ ID NO.3 was mutated to isoleucine, the glycine at position 164 was mutated to threonine, and the glycine at position 208 was mutated to proline.
4. A gene encoding the nitrile hydratase mutant according to any one of claims 1 to 3.
5. A recombinant microbial cell expressing the nitrile hydratase mutant according to any one of claims 1 to 3.
6. A recombinant Escherichia coli, characterized in that BL21 (DE3) is used as a host and pET24a is used as a vector to express the nitrile hydratase mutant.
7. A method for improving the catalytic activity and / or thermal stability of nitrile hydratase, characterized in that: The α subunit of nitrile hydratase is subjected to one or more mutations of T2M, G32D, G86F, K57F, E183F, and / or the β subunit of nitrile hydratase is subjected to one or more mutations of T149I, T149H, R160W, G164T, G164M, G208P, A230V, A230R.
8. The method according to claim 7, characterized in that The α subunit of the nitrile hydratase has an amino acid sequence shown in SEQ ID NO.1, and the β subunit of the nitrile hydratase has an amino acid sequence shown in SEQ ID NO.
3.
9. A method for preparing the mutant according to any one of claims 1 to 3, characterized in that: The recombinant Escherichia coli of claim 6 is cultured in a culture medium for a period of time, and IPTG and Co 2+ Induce enzyme expression.
10. Use of the nitrile hydratase mutant according to any one of claims 1 to 3, or the gene according to claim 4, or the recombinant microbial cell according to claim 6, or the recombinant Escherichia coli according to claim 6 in the preparation of amide compounds.