Modification of the amino acid motif of a nitrile hydratase substrate channel for the preparation of cinnamamide
By modifying the amino acids of the substrate channel of pseudocarcinoma thermophila nitrile hydratase, a nitrile hydratase mutant with improved catalytic activity was constructed, which solved the problem of low catalytic efficiency of existing nitrile hydratase on larger sterically hindered substrates, and achieved a significant effect of improving enzyme activity.
Patent Information
- Application Number
- CN202211369760.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-11-03
- Publication Date
- 2025-03-04
- Estimated Expiration
- 2042-11-03
AI Technical Summary
The existing nitrile hydratases have problems such as poor stability, low catalytic activity and narrow substrate spectrum, especially the catalytic efficiency of substrates with large steric hindrances such as cinnamon nitrile.
By modifying the substrate channel amino acids of nitrile hydratase derived from Pseudocarcinothermia, specifically mutating the amino acids in phenylalanine at the 37th position, leucine at the 48th position or glutamine at the α subunit, a nitrile hydratase mutant with improved catalytic activity was constructed.
The enzyme activity of the mutant is significantly improved, the catalytic efficiency of cinnamon nitrile is increased by more than 10 times, broadening the substrate spectrum of nitrile hydratase, and has the potential to be used in the preparation of cinnamide in industrial production.
Smart Images
Figure CN115772514B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the modification of amino acid motif of nitrile hydratase substrate channel for the preparation of cinnamamide, belonging to the technical field of bioengineering. Background Art
[0002] Amide substances carrying unique amide groups have special physiological functions and potential biological activities. They are advantageous resources for synthesizing a large number of fine chemicals, polymers and synthetic intermediates. At present, many synthetic methods have been developed for the preparation of amide substances. The synthesis of cinnamamide and its derivatives is a classic case. They can be used as carrier scaffolds for many natural products. For example, such scaffolds can be used as effective templates for the design and production of new drug molecules with pharmacological activity. At present, the acquisition of cinnamamide and its derivatives in industrial production is mainly based on traditional chemical synthesis, and there are very few literature reports on bioenzymatic catalysis. In the chemical production process, the operation is relatively cumbersome and requires extreme temperatures, strong bases, toxic reagents such as triethylamine, etc. These harsh reaction conditions have low safety factors, which are inconsistent with the concept of green production and urgently need to be improved.
[0003] Nitriles are valuable intermediates in synthesis, and they can be easily obtained from many simple, direct and cost-effective synthetic methods. Nitrile hydratase (NHase, EC4.2.1.84) is a metalloenzyme that can catalyze the conversion of nitrile substances into high-value-added amide compounds through hydration reactions. It has been widely used in the industrial production of nicotinamide and acrylamide. At present, the biological production technology of amides is gradually replacing the traditional chemical method with its advantages of green environmental protection, mild reaction conditions and high safety factor, which is in line with the concept of sustainable development and green production.
[0004] Nitrile hydratase is usually composed of two subunits, α and β. Studies have found that most of the reported nitrile hydratases from prokaryotes have problems such as poor stability, low catalytic activity, and a narrow catalytic substrate spectrum. Many studies have also attempted to modify it to improve its related properties. However, the current low catalytic efficiency and narrow substrate spectrum still limit the further development and application of nitrile hydratase. Especially for substrates with large steric hindrance such as cinnamonitrile, the activity is even lower. Summary of the invention
[0005] In view of the existing technical difficulties and problems, the present invention transforms the substrate channel amino acids of nitrile hydratase from Pseudonocardia thermophila in order to broaden the substrate spectrum and improve the catalytic performance.
[0006] The invention provides a nitrile hydratase mutant. The nitrile hydratase comprises an alpha subunit and a beta subunit, and a nitrile hydratase PtNHase derived from Pseudonocardia thermophila is used as a parent. The amino acid sequence of the alpha subunit of the nitrile hydratase parent is shown in SEQ ID NO.1, and the amino acid sequence of the beta subunit is shown in SEQ ID NO.2. The mutant is prepared by mutating at least one of the 37th phenylalanine of the beta subunit, the 48th leucine of the beta subunit, or the 89th glutamine of the alpha subunit.
[0007] In one embodiment, the mutant is any one of the following (a) to (e):
[0008] (a) The mutant is a mutant in which the phenylalanine at position 37 and the leucine at position 48 of the β subunit are mutated and replaced;
[0009] (b) The mutant is a mutant in which the glutamine at position 89 of the α subunit is mutated and replaced;
[0010] (c) The mutant is a mutant in which phenylalanine at position 37 and leucine at position 48 of the β subunit and glutamine at position 89 of the α subunit are mutated and substituted;
[0011] (d) The mutant is a mutant in which the phenylalanine at position 37 of the β subunit and the glutamine at position 89 of the α subunit are mutated and substituted;
[0012] (e) The mutant is a mutant in which the leucine at position 48 of the β subunit and the glutamine at position 89 of the α subunit are mutated and substituted.
[0013] In one embodiment, the mutant is a mutant in which position 37 of the β subunit is mutated to proline or valine, and position 48 is mutated to alanine, cysteine, glutamic acid, glycine, histidine, lysine, proline, arginine or threonine.
[0014] In one embodiment, the mutant is a mutant in which position 37 of the β subunit is mutated to proline, and position 48 is mutated to alanine, proline or threonine.
[0015] In one embodiment, the mutant is a mutation in which position 89 of the α subunit is mutated to cysteine, aspartic acid, lysine, asparagine, serine, or valine.
[0016] In one embodiment, the mutant is a mutation in which position 89 of the α subunit is mutated to asparagine.
[0017] The present invention provides a gene encoding the mutant.
[0018] The invention provides a recombinant plasmid carrying the gene.
[0019] In one embodiment, the recombinant plasmid uses the pET series as an expression vector.
[0020] In one embodiment, the recombinant plasmid uses pET-24a(+) as an expression vector.
[0021] The present invention provides a host cell expressing the mutant or containing the gene.
[0022] In one embodiment, the host cell comprises a prokaryotic or eukaryotic microorganism.
[0023] In one embodiment, the host cell is Escherichia coli.
[0024] In one embodiment, the host cell is E. coli BL21 (DE3).
[0025] The invention provides a recombinant strain, which expresses the nitrile hydratase mutant.
[0026] In one embodiment, the recombinant strain uses Escherichia coli BL21 (DE3) as a host and pET-24a (+) as an expression vector.
[0027] The invention provides a method for improving the catalytic activity of nitrile hydratase. The method comprises the following steps: using nitrile hydratase PtNHase derived from Pseudonocardia thermophila as a parent; the amino acid sequence of the α subunit of the nitrile hydratase parent is shown in SEQ ID NO.1, and the amino acid sequence of the β subunit is shown in SEQ ID NO.2; and the mutant comprises at least one of phenylalanine at position 37 of the β subunit, leucine at position 48 of the β subunit, or glutamine at position 89 of the α subunit being mutated.
[0028] In one embodiment, the method is any one of the following (a) to (e):
[0029] (a) The method comprises the following steps: mutating and replacing the phenylalanine at position 37 and the leucine at position 48 of the β subunit;
[0030] (b) The method comprises the step of mutating the glutamine at position 89 of the α subunit to cause substitution;
[0031] (c) The method comprises mutating and replacing phenylalanine at position 37 and leucine at position 48 of the β subunit and glutamine at position 89 of the α subunit;
[0032] (d) The method comprises the steps of mutating the phenylalanine at position 37 of the β subunit and the glutamine at position 89 of the α subunit;
[0033] (e) The method comprises mutating and replacing the leucine at position 48 of the β subunit and the glutamine at position 89 of the α subunit.
[0034] In one embodiment, the method is to mutate position 37 of the β subunit to proline or valine, and position 48 to alanine, cysteine, glutamic acid, glycine, histidine, lysine, proline, arginine or threonine.
[0035] In one embodiment, the method is to mutate position 37 of the β subunit to proline, and position 48 to alanine, proline or threonine.
[0036] In one embodiment, the method is to mutate position 89 of the α subunit to cysteine, aspartic acid, lysine, asparagine, serine, or valine.
[0037] In one embodiment, the method is to mutate position 89 of the α subunit to asparagine.
[0038] The invention provides a method for preparing cinnamamide. The method takes cinnamonitrile as a substrate and utilizes the nitrile hydratase mutant to catalyze and generate cinnamamide.
[0039] The present invention also provides application of the nitrile hydratase mutant, or the gene, or the recombinant plasmid, or the host cell in preparing cinnamamide.
[0040] In one embodiment, the genes on the expression vector are connected in the order of the gene encoding the β subunit, the gene encoding the α subunit, and the gene encoding the regulatory protein.
[0041] Beneficial effects:
[0042] The present invention finds that the amino acid residues at positions 37 and 48 on the β subunit and position 89 on the α subunit of Pt-NHase may be in the key structural domain of nitrile hydratase, and play an important role in the catalytic activity of nitrile hydratase. The present invention provides a Pt-NHase mutant, wherein the mutant is mutated to phenylalanine and leucine at positions 37 and 48 on the β subunit relative to the wild type, to obtain mutants F37P-L48A, F37P-L48P, and F37P-L48T; and glutamine at position 89 on the α subunit is mutated to obtain mutant Q89N. For cinnamonitrile catalysis, the enzyme activities of the mutants F37P-L48A, F37P-L48P, F37P-L48T and Q89N in the present invention are significantly improved compared with the wild enzyme, with the highest improvement being more than 10 times, which opens up the further development and application of nitrile hydratase and has great potential in the industrial production of cinnamamide. BRIEF DESCRIPTION OF THE DRAWINGS
[0043] Figure 1 Preliminary screening of a mutant library constructed for the three amino acid residues of the PtNHase substrate channel and enzyme activity determination.
[0044] Figure 2 To screen the mutant library of PtNHase and determine the enzyme activity. DETAILED DESCRIPTION
[0045] (I) Culture medium
[0046] LB medium: tryptone 10.0 g / L, yeast extract 5.0 g / L, NaCl 10.0 g / L, kanamycin final concentration 50 μg / mL.
[0047] 2×YT medium: tryptone 16.0 g / L, yeast extract 10.0 g / L, NaCl 5.0 g / L, kanamycin final concentration 50 μg / mL.
[0048] (II) Enzyme activity detection method
[0049] Enzyme activity of nitrile hydratase (U): Unit enzyme activity is defined as the amount of enzyme required to catalyze the conversion of nitrile substrates to 1 μmol of amide product per minute at 25°C.
[0050] Nitrile hydratase activity (U / mL): the enzyme activity of nitrile hydratase per milliliter.
[0051] Example 1: Construction of various mutants of Pt NHase
[0052] The gene encoding the β subunit (amino acid sequence as shown in SEQ ID NO.2), the gene encoding the α subunit (amino acid sequence as shown in SEQ ID NO.1) and the gene encoding the regulatory protein were sequentially connected to the restriction sites NdeI (CATATG) and BamHI (GGATCC) of the expression vector pET-24a(+), and transformed into E. coli JM109. After culturing on LB plates, single clones were picked and sequenced and verified by Anshengda (Suzhou) Biotechnology Co., Ltd. to obtain positive transformants. Plasmids were extracted from the positive transformants, namely the wild-type plasmid pET24a(+)-PtNHase WT.
[0053] First, the wild-type plasmid pET24a(+)-PtNHase WT was used as a template, the mutant sequence was designed on the primers, and the DNA fragment with the mutated base sequence was amplified by PCR. The corresponding primer sequences used for different mutant plasmids are shown in Table 1. The whole plasmid PCR method was used to construct the single point mutant plasmids pET24a(+)-F37P, pET24a(+)-L48A, pET24a(+)-L48P, pET24a(+)-L48T and pET24a(+)-Q89N. Then, the single point mutation plasmids were used as templates to construct mutant plasmids pET24a(+)-F37P-L48A, pET24a(+)-F37P-L48P, and pET24a(+)-F37P-L48T.
[0054] The amplification system is shown in Table 2. The PCR amplification reaction conditions are pre-denaturation at 98°C for 1 min, denaturation at 98°C for 10 s, annealing at 55°C for 15 s, extension at 72°C for 1 min30 s, and extension at 72°C for 5 min, for a total of 30 cycles.
[0055] Table 1 Primer sequences
[0056]
[0057]
[0058] Table 2 PCR amplification system
[0059]
[0060] Example 2: Catalytic efficiency of wild-type and mutant Pt NHase for cinnamonitrile
[0061] The wild-type plasmid pET24a(+)-PtNHaseWT and mutant plasmids pET24a(+)-F37P-L48A, pET24a(+)-F37P-L48P, pET24a(+)-F37P-L48T and pET24a(+)-Q89N in Example 1 were transformed into E. coli BL21 (DE3), and single colonies were picked to 5 mL LB medium and cultured at 37°C and 200 rpm for 7-8 h. The seed solution was transferred to 100 mL 2×YT medium at 1% (v / v) and cultured at 37°C and 200 rpm until OD 600 to 0.6-0.8, add isopropylthiogalactoside (IPTG) with a final concentration of 0.4 mM and 0.1 g / L CoCl 2 6H 2O, change the culture temperature to 25℃, induce expression for 16h, and centrifuge to obtain wild-type WT and mutant cells F37P-L48A, F37P-L48P, F37P-L48T and Q89N.
[0062] Use 10mM KPB (press K 2 HPO 4 :KH 2 PO 4 =4:1, pH 7.4) buffer was used to adjust the OD of WT and F37P-L48A, F37P-L48P, F37P-L48T and Q89N mutant cells. 600 Adjust to 0.5, take 10 μL to a 1.5 mL centrifuge tube, and place it on a 25°C metal bath. Add 490 μL of substrate (5 mM cinnamonitrile solution, containing 10% methanol) to the centrifuge tube, vortex thoroughly, react at 25°C for 5 minutes, and then add 500 μL of pure acetonitrile solution to terminate. Pass the reaction solution through a 0.22 μm filter membrane for liquid phase detection.
[0063] Liquid phase detection method: the mobile phase composition is acetonitrile: water = 1:2 (v / v), the flow rate is 1 mL / min, the detection wavelength is 261 nm, the column temperature is 40°C, and the amount of cinnamamide produced in the reaction system is determined.
[0064] The enzyme activity calculation results of WT and each mutant are shown in Figure 1 and 2 As shown, the enzyme activity of the wild enzyme WT was 5 U / mL, and the enzyme activities of the mutants F37P-L48A, F37P-L48P, F37P-L48T and Q89N were 53.6 U / mL, 50.1 U / mL, 46.5 U / mL and 47.0 U / mL, respectively.
[0065] That is, when the amino acid residues at positions 37 and 48 on the above-mentioned β subunit mutate at the same time, or the amino acid residue at position 89 on the α subunit mutates, the enzymatic activity of nitrile hydratase is significantly improved, indicating that the above-mentioned amino acid residues at positions 37, 48 and 89 may be in the key structural domain of the enzyme and play an important role in the catalytic activity of nitrile hydratase.
[0066] Table 3 Comparison of enzyme activity of wild type and mutants on cinnamonitrile
[0067] sample WT F37P-L48A F37P-L48P F37P-L48T Q89N Enzyme activity (U / mL) 5.0±1.0 53.6±2.7 50.1±3.0 46.5±2.5 47.0±2.1
[0068] Comparative Example 1
[0069] For a specific implementation, see the embodiment. The difference is that a single-point saturation mutation is performed on the wild-type PtNHase, and the phenylalanine at position 37 and the leucine at position 48 on the β subunit and the glutamine at position 89 on the α subunit are replaced by the other 19 amino acids (mutated to A / C / D / E / F / G / H / I / K / L / M / N / P / Q / R / S / T / V / W / Y, respectively). After the mutant is cultured and induced to express, the cells are adjusted to the same OD of 0.5, and the enzyme activity is measured. The results show that when position 37 is mutated to proline, position 48 is mutated to alanine, cysteine, histidine, lysine, proline, arginine and threonine, and position 89 is mutated to aspartic acid, asparagine, serine and valine, the enzyme activity is higher ( Figure 1 ).
[0070] Table 4 Comparison of the catalytic activity of wild-type and mutant whole cells towards cinnamonitrile
[0071] sample Enzyme activity (U / mL) sample Enzyme activity (U / mL) sample Enzyme activity (U / mL) WT 5.0±1.0 — — — — F37A 5.0±1.0 L48A 28.1±2.5 Q89A 15.9±1.8 F37C 6.3±0.8 L48C 26.6±2.3 Q89C 25.5±2.0 F37D 6.4±1.0 L48D 13.9±1.9 Q89D 39.8±3.9 F37E 6.9±0.9 L48E 13.9±1.6 Q89E 29.9±3.0 F37G 9.8±1.2 L48F 19.7±2.1 Q89F 12.4±1.0 F37H 3.8±0.5 L48G 22.2±2.4 Q89G 20.4±2.0 F37I 10.0±1.0 L48H 25.6±2.1 Q89 — F37K 1.5±0.3 L48I 8.5±1.8 Q89I — F37L 8.5±1.2 L48K 27.9±2.6 Q89K 28.2±2.5 F37M 1.0±0.1 L48M — Q89L 29.4±3.0 F37N 8.2±1.5 L48N 21.2±1.4 Q89M 2.0±0.2 F37P 12.0±2.0 L48P 32.6±2.6 Q89N 47.0±4.5 F37Q 8.2±1.3 L48Q 19.7±2.0 Q89P — F37R 0.5±0.1 L48R 35.0±3.1 Q89R 20.7±1.9 F37S 6.3±1.1 L48S 11.2±2.1 Q89S 30.5±3.0 F37T 8.1±1.4 L48T 21.7±1.8 Q89T 14.4±1.4 F37V 10.6±1.7 L48V 21.8±1.9 Q89V 43.1±4.3 F37W 3.4±0.7 L48W 9.3±1.4 Q89W 10.7±1.1 F37Y 0.8±0.1 L48Y 17.4±2.0 Q89 1.5±0.2
[0072] Comparative Example 2
[0073] Specific implementation method See the embodiment and comparative example 1, the difference is that the second round of PCR is performed on the single point mutants with higher activity in the comparative example 1 to obtain mutants combined in pairs. The results show that the enzyme activity of F37P-L48A, F37P-L48H, F37P-L48P, F37P-L48T and Q89N-L48T is higher ( Figure 2 ).
[0074] Table 5 Comparison of the catalytic activity of wild-type and mutant whole cells towards cinnamonitrile
[0075] sample Enzyme activity (U / mL) sample Enzyme activity (U / mL) WT 5.0±1.0 — — F37P-L48A 55.7±4.0 Q89D-L48A 23.8±2.2 F37P-L48C 39.8±4.0 Q89D-L48H 13.4±1.3 F37P-L48H 36.9±4.2 Q89D-L48P 23.5±2.5 F37P-L48K 17.5±1.5 Q89D-L48T 23.8±2.4 F37P-L48P 46.5±2.9 Q89N-L48A 28.9±3.0 F37P-L48R 39.2±4.0 Q89N-L48H 5.1±0.9 F37P-L48T 47.2±4.5 Q89N-L48P 30.5±3.0 F37P-Q89D 25.9±2.3 Q89N-L48T 46.4±4.6 F37P-Q89N 29.8±3.0 Q89S-L48A 17.0±1.7 F37P-Q89S 29.5±3.0 Q89S-L48H 14.5±1.5 F37P-Q89V 26.0±2.7 Q89S-L48P 11.6±1.2 Q89S-L48T 22.3±2.3 Q89V-L48A 25.3±2.6 Q89V-L48H 3.8±0.5 Q89V-L48P 23.2±2.6 Q89V-L48T 23.9±2.4
[0076] Comparative Example 3
[0077] Specific implementation method See the embodiment and comparative examples 1 and 2, the difference is that the combination mutants F37P-L48A, F37P-L48P, and F37P-L48T with higher activity in the above comparative example 2 are used as templates, respectively, and the third round of PCR is carried out, and it is iterated with the single point mutants Q89D, Q89N, Q89S, and Q89V to obtain 12 three-point combination mutants. The results show that the enzyme activities of F37P-L48T-Q89S and F37P-L48T-Q89N are relatively high, which are 23.3 and 15.9 U / mL, respectively. However, compared with the enzyme activities of the four mutants F37P-L48A, F37P-L48P, F37P-L48T, and Q89N in the embodiment, they are still significantly reduced ( Figure 2 ).
[0078] Table 6 Comparison of the catalytic activity of wild-type and mutant whole cells towards cinnamonitrile
[0079]
[0080]
[0081] 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, characterized in that The nitrile hydratase comprises an α subunit and a β subunit, and the nitrile hydratase PtNHase derived from Pseudonocardia thermophila is used as a parent; the amino acid sequence of the α subunit of the nitrile hydratase parent is shown in SEQ ID NO.1, and the amino acid sequence of the β subunit is shown in SEQ ID NO.2; The mutant is a mutant in which the phenylalanine at position 37 of the β subunit is mutated to proline; or, mutating the phenylalanine at position 37 of the β subunit to proline, and mutating the leucine at position 48 to alanine; or, mutating the phenylalanine at position 37 of the β subunit to proline, and mutating the leucine at position 48 to cysteine; or, mutating the phenylalanine at position 37 of the β subunit to proline, and mutating the leucine at position 48 to histidine; or, mutating the phenylalanine at position 37 of the β subunit to proline, and mutating the leucine at position 48 to lysine; or, mutating the phenylalanine at position 37 of the β subunit to proline, and mutating the leucine at position 48 to proline; or, mutating the phenylalanine at position 37 of the β subunit to proline, and mutating the leucine at position 48 to arginine; or, mutating the phenylalanine at position 37 of the β subunit to proline, and mutating the leucine at position 48 to threonine; or, mutating the phenylalanine at position 37 of the β subunit to proline and mutating the glutamine at position 89 to aspartic acid; or, mutating the phenylalanine at position 37 of the β subunit to proline, and mutating the glutamine at position 89 to asparagine; or, mutating the phenylalanine at position 37 of the β subunit to proline and the glutamine at position 89 to serine; or, mutating the phenylalanine at position 37 of the β subunit to proline, and mutating the glutamine at position 89 to valine; or, mutating the phenylalanine at position 37 of the β subunit to proline, the leucine at position 48 to alanine, and the glutamine at position 89 to aspartic acid; or, mutating the phenylalanine at position 37 of the β subunit to proline, the leucine at position 48 to alanine, and the glutamine at position 89 to asparagine; or, mutating the phenylalanine at position 37 of the β subunit to proline, the leucine at position 48 to alanine, and the glutamine at position 89 to serine; or, mutating the phenylalanine at position 37 of the β subunit to proline, the leucine at position 48 to alanine, and the glutamine at position 89 to valine; or, mutating the phenylalanine at position 37 of the β subunit to proline, mutating the leucine at position 48 to proline, and mutating the glutamine at position 89 to valine; or, mutating the phenylalanine at position 37 of the β subunit to proline, the leucine at position 48 to threonine, and the glutamine at position 89 to asparagine; or, mutating the phenylalanine at position 37 of the β subunit to proline, the leucine at position 48 to threonine, and the glutamine at position 89 to serine; Alternatively, the phenylalanine at position 37 of the β subunit is mutated to proline, the leucine at position 48 is mutated to threonine, and the glutamine at position 89 is mutated to valine.
2. A gene encoding the mutant according to claim 1.
3. A recombinant plasmid carrying the gene according to claim 2.
4. A host cell expressing the mutant according to claim 1, or containing the gene according to claim 2.
5. A recombinant strain, characterized in that: Expressing the nitrile hydratase mutant according to claim 1.
6. A method for improving the catalytic activity of nitrile hydratase, characterized in that: The method comprises the following steps: using a nitrile hydratase PtNHase derived from Pseudonocardia thermophila as a parent for mutation; the amino acid sequence of the α subunit of the nitrile hydratase parent is shown in SEQ ID NO.1, and the amino acid sequence of the β subunit is shown in SEQ ID NO.2; and the mutation comprises mutating the 37th phenylalanine of the β subunit to proline; or, mutating the phenylalanine at position 37 of the β subunit to proline, and mutating the leucine at position 48 to alanine; or, mutating the phenylalanine at position 37 of the β subunit to proline, and mutating the leucine at position 48 to cysteine; or, mutating the phenylalanine at position 37 of the β subunit to proline, and mutating the leucine at position 48 to histidine; or, mutating the phenylalanine at position 37 of the β subunit to proline, and mutating the leucine at position 48 to lysine; or, mutating the phenylalanine at position 37 of the β subunit to proline, and mutating the leucine at position 48 to proline; or, mutating the phenylalanine at position 37 of the β subunit to proline, and mutating the leucine at position 48 to arginine; or, mutating the phenylalanine at position 37 of the β subunit to proline, and mutating the leucine at position 48 to threonine; or, mutating the phenylalanine at position 37 of the β subunit to proline and mutating the glutamine at position 89 to aspartic acid; or, mutating the phenylalanine at position 37 of the β subunit to proline, and mutating the glutamine at position 89 to asparagine; or, mutating the phenylalanine at position 37 of the β subunit to proline and the glutamine at position 89 to serine; or, mutating the phenylalanine at position 37 of the β subunit to proline, and mutating the glutamine at position 89 to valine; or, mutating the phenylalanine at position 37 of the β subunit to proline, the leucine at position 48 to alanine, and the glutamine at position 89 to aspartic acid; or, mutating the phenylalanine at position 37 of the β subunit to proline, the leucine at position 48 to alanine, and the glutamine at position 89 to asparagine; or, mutating the phenylalanine at position 37 of the β subunit to proline, the leucine at position 48 to alanine, and the glutamine at position 89 to serine; or, mutating the phenylalanine at position 37 of the β subunit to proline, the leucine at position 48 to alanine, and the glutamine at position 89 to valine; or, mutating the phenylalanine at position 37 of the β subunit to proline, mutating the leucine at position 48 to proline, and mutating the glutamine at position 89 to valine; or, mutating the phenylalanine at position 37 of the β subunit to proline, the leucine at position 48 to threonine, and the glutamine at position 89 to asparagine; or, mutating the phenylalanine at position 37 of the β subunit to proline, the leucine at position 48 to threonine, and the glutamine at position 89 to serine; Alternatively, the phenylalanine at position 37 of the β subunit is mutated to proline, the leucine at position 48 is mutated to threonine, and the glutamine at position 89 is mutated to valine.
7. A method for producing cinnamamide, characterized in that: Cinnamonitrile is used as a substrate and cinnamamide is produced by catalysis using the mutant described in claim 1 or the recombinant strain described in claim 5.
8. Use of the mutant according to claim 1, or the gene according to claim 2, or the recombinant plasmid according to claim 3, or the host cell according to claim 4, or the recombinant strain according to claim 5 in the preparation of cinnamamide.