Phenylalanine lyase mutant and preparation method of alpha-methyl amino acid

Abstract

The invention provides a phenylalanine lyase mutant and a preparation method of alpha-methyl amino acid. Wherein the phenylalanine lyase mutant comprises (a) a protein mutated on the basis of wild type phenylalanine lyase as shown in SEQ ID NO: 1, and the mutation is selected from mutation of any one or more of the following sites: N438, C288, Y304, F90, P317, S114 and the like; or (b) a protein which has more than 70% of homology with the amino acid sequence limited in (a) and has phenylalanine lyase activity. The method can solve the problem that non-natural amino acid is difficult to simply and efficiently synthesize in the prior art and is suitable for the field of enzyme catalysis.

Description

Technical Field

[0001] This invention relates to the field of enzyme catalysis, and more specifically, to a method for preparing a phenylalanine lyase mutant and α-methyl amino acids. Background Technology

[0002] Non-natural amino acids, due to their unique physical, chemical, and biological properties, show broad application prospects in fields such as biomedicine, basic research, materials science, and synthetic biology. As key components of many bioactive molecules and drugs, non-natural amino acids can effectively enhance the stability of peptide chains and proteins and improve their resistance to chemical and enzymatic degradation.

[0003] Currently, the main method for synthesizing non-natural amino acids is chemical synthesis. However, chemical synthesis suffers from drawbacks such as low yields of non-natural amino acids, the use of toxic reagents like sodium cyanide, cumbersome processes for preparing chiral precursors, air sensitivity of organometallic catalysts, and relatively expensive chiral ligands. Therefore, developing synthetic methods for non-natural amino acids using inexpensive and readily available raw materials, with simple and easy-to-operate reaction systems, has become a key scientific issue linking basic research and industrial applications, and possesses enormous market demand. Summary of the Invention

[0004] The main objective of this invention is to provide a method for preparing phenylalanine lyase mutants and α-methyl amino acids, thereby solving the problem of the difficulty in synthesizing non-natural amino acids simply and efficiently in the prior art.

[0005] To achieve the above objectives, according to a first aspect of the present invention, a phenylalanine lyase mutant is provided, comprising: (a) a protein mutated based on the wild-type phenylalanine lyase shown in SEQ ID NO: 1, wherein the mutation is selected from mutations at any one or more of the following sites: N438, C288, Y304, F90, P317, S114, M354, V293, C103, V59, T89, F77, G46, Q108, M173, R295, I379, I332, I111, V303, T110, Y145, Y357, I60, I63, Y437, R 144, N288, R289, H290, Q291, E302, S081, V115, K117, L113, I230, A370, H243, A378, I382, M383, T399, S81, S192, E82, E197, S56, Q85, K407, L276, K406 or Q273; or (b) a protein that has more than 70% homology with the amino acid sequence defined in (a) and has phenylalanine lyase activity.

[0006] Further, in (a) above, the mutation is selected from any one or more of the following mutations: N438H, C288R, C288A, C288F, C288N, C288T, C288V, Y304L, Y304C, Y304V, F90Y, P317F, P317M, S114A, S114N, S114C, M354H, V293I, C103E, C103S, V59L, T89V, F77D, F77I, F77S, F77Y, F77N, G46C, Q108M, M173R, R295Q, R295K, I3 79L, I332A, I332D, I332S, I332N, I332H, I332Q, I111S, I111T, V303E, T110G, Y145F, Y3 57H, I60M, I63L, I63V, Y437F, R144Y, R144V, N288V, R289I, H290L, H290I, Q291N, E302W , S081R, V115I, K117V, L113V, I230A, A370G, H243Y, A378Q, I382M, M383L, T399S, T399C The series of amino acids are: S81R, S81E, S81K, S192D, E82K, E197D, S56A, Q85R, K407T, L276E, L276T, L276N, L276D, L276K, L276R, L276Y, K406L, K406M, Q273A, or Q273S. The letters preceding the numbers represent the original amino acids, and the letters following the numbers represent the mutated amino acids.

[0007] Furthermore, the above mutations include any one of the following amino acid mutations: N438H, N438H+C288R, N438H+Y304C, N438H+Y304V, N438H+Y304L, N438H+P317F, N438H+P317M, N438H+I379L, N438H+F90Y, N438H+C288R+S114A, N438H+C288R+S114N, N438H+C288R+M354H, N438H+C288R+V293I, N438H+C288R+C103E, N438H+C288R+V59L, N438H+C288R+ T89V, N438H+C288R+F77D, N438H+C288R+G46C, N438H+C288R+Q108M, N43 8H+C288R+Y304L, N438H+C288R+C103S, N438H+S114A+C288A, N438H+S114 A+C288F, N438H+S114A+C288N, N438H+S114A+C288T, N438H+S114A+C288V , N438H+S114A+C288T+M173R, N438H+S114A+C288T+Q108M, N438H+C288R+ S114A+M354H, N438H+C288R+S114A+C103E, N438H+C288R+S114A+T89V, N 438H+C288R+Y304L+F90Y, N438H+F77Y+S114C+C288N+R295Q, N438H+77Y+ S114A+C288T+R295K, N438H+F77S+S114A+C288N+R295Q, N438H+F77S+S11 4A+C288N+R295K, N438H+F77Y+S114C+C288N+R295Q+V59L, N438H+F77I+S 114C+C288N+R295Q, N438H+F77S+S114C+C288N+R295Q, N438H+F77Y+S114 C+C288N+R295Q, N438H+F77Y+S114C+C288N+R295Q+I332D, N438H+F77Y+S 114C+C288N+R295Q+I111S, N438H+F77Y+S114C+C288N+R295Q+I111T, N43 8H+F77Y+S114C+C288N+R295Q+V303E, N438H+F77N+S114C+C288N+R295Q,N438H+F77Y+S114C+C288N+R295Q+I332S、N438H+F77Y+S114C+C288N+R295Q+T110G、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S、N438H+F77Y+S114C+C288N+R295Q+V303E+Y145F、N438H+F77Y+S114C+C288N+R295Q+V303E+I332N、N438H+F77Y+S114C+C288N+R295Q+V303E+Y357H、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I63L、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+Y437F、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I332N、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I332H、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+R144Y、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Y145F+I332N、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+I63V、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+R144V、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+N288V、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+R289I、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+H290L、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+E302W、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+I332N、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+S081R、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+K117V、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+I230A、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+A370G、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+H243Y、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+A378Q、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+I332Q、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+I382M、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T110G、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+M383L、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399S、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+S81R、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+H290I、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+S192D、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+E82K、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+E197D、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+E197D+I332A、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+E197D+S56A、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+S81E+Q85R+H243Y+I332N、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+S81K+Q85K+H243Y+I332N、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T+L276E、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T+L276T、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T+L276N, N438H+F77Y+S1 14C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T+L276D, N438H+F77Y+S114C+C288N+R295 Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T+L276K, N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+ I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T+L276Y, N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V11 5I+L113V+T399C+E302W+I332D+K407T+K406L, N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C +E302W+I332D+K407T+K406M, N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K 407T+L276R, N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T+Q273A, N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T+Q273S, C288R, Y304L or F90Y.

[0008] To achieve the above objectives, according to a second aspect of the present invention, a DNA molecule is provided that encodes the aforementioned phenylalanine lyase mutant.

[0009] To achieve the above objectives, according to a third aspect of the present invention, a recombinant plasmid is provided, the recombinant plasmid containing the aforementioned DNA molecule.

[0010] To achieve the above objectives, according to a fourth aspect of the present invention, a host cell is provided, wherein the host cell contains the aforementioned DNA molecule or the aforementioned recombinant plasmid.

[0011] Furthermore, the host cells mentioned above include Escherichia coli, which may be selected as Escherichia coli BL21.

[0012] To achieve the above objective, according to a fifth aspect of the present invention, a method for preparing α-methyl amino acids is provided, the method comprising: using the above-mentioned phenylalanine lyase to catalyze the reaction of an olefinic acid substrate and an amino donor to prepare the above-mentioned α-methyl amino acids.

[0013] Furthermore, the above-mentioned olefinic acid substrate is The corresponding α-methyl amino acid is ; wherein R is selected from substituted or unsubstituted aryl, heteroaryl or fused ring, and the number of carbon atoms in the aryl, heteroaryl or fused ring is 4-10.

[0014] Furthermore, the aforementioned fused rings include quinolinyl, benzimidazolyl, benzofuranyl, benzothiopheneyl, isobenzofuranyl, dibenzofuranyl, dibenzothiopheneyl, benzothiazolyl, benzoisothiazolyl, benzoisooxazolyl, isoquinolinyl, cenolinyl, quinazolinyl, quinoxolinyl, carbazole, phenanthroxazolyl, phenadiazolyl, benzodiazepine, and dihydroacridyl.

[0015] Furthermore, the heteroatoms in the aforementioned heteroaryl group are selected from one or more of nitrogen, oxygen, or sulfur.

[0016] Furthermore, the above substitutions include one or more hydrogen atoms in the aryl, heteroaryl, or fused ring being independently substituted by substituents, which include one or more of halogens, alkyl groups, nitro groups, trifluoromethyl groups, phenyl groups, hydroxyl groups, or alkoxy groups.

[0017] Furthermore, the above-mentioned olefinic acid substrate is selected from any one or more of the following: , , , , , , , , , , , , , , , or .

[0018] Furthermore, the aforementioned amino donor includes one or more of ammonium carbamate, ammonium chloride, ammonium sulfate, ammonia, or ammonium carbonate.

[0019] Furthermore, the above reactions are carried out in an aqueous system or a two-phase system.

[0020] Furthermore, the temperature of the above reaction is 10-50℃.

[0021] Furthermore, the temperature of the above reaction is 25-45℃.

[0022] To achieve the above objectives, according to a sixth aspect of the present invention, the application of the above-described phenylalanine lyase mutant, the above-described DNA molecule, the above-described recombinant plasmid, the above-described host cell, or the above-described preparation method in the preparation of α-methyl amino acids is provided.

[0023] Furthermore, the above-mentioned α-methyl amino acids include those with... Amino acids in the structure.

[0024] By applying the technical solution of this invention, the above-mentioned phenylalanine lyase mutant can efficiently catalyze the binding of enoic acid substrates and amino donors to prepare α-methyl amino acids. Compared with the wild-type enzyme, the above-mentioned phenylalanine lyase mutant has a higher conversion rate and efficiency in the preparation of α-methyl amino acids, which highly meets the urgent needs of modern pharmaceutical industry for green, efficient and safe production. Attached Figure Description

[0025] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0026] Figure 1 A schematic diagram of a catalytic reaction according to an embodiment of the present invention is shown.

[0027] Figure 2 A schematic diagram of the chemical reaction for the preparation of (S)-2-amino-2-methyl-3-phenylalanine by catalytic 2-methyl-3-phenylacrylic acid according to Example 2 of the present invention is shown. Detailed Implementation

[0028] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0029] As mentioned in the background section, it is difficult to synthesize non-natural amino acids using existing technologies, which cannot meet the requirements of industrial production. Therefore, in this application, the inventors attempted to develop a phenylalanine lyase mutant that promotes the synthesis of non-natural amino acids. Through directed evolution, a phenylalanine lyase mutant was obtained, which can efficiently prepare the target product α-methyl amino acid with 100% atom economy and excellent stereoselectivity. Its advantages are unmatched by traditional chemical methods and highly meet the urgent needs of the modern pharmaceutical industry for green, efficient, and safe production. Therefore, a series of protection schemes in this application are proposed.

[0030] In a first typical embodiment of this application, a phenylalanine lyase mutant is provided, comprising: (a) a protein mutated based on the wild-type phenylalanine lyase shown in SEQ ID NO: 1, wherein the mutation is selected from mutations at any one or more of the following sites: N438, C288, Y304, F90, P317, S114, M354, V293, C103, V59, T89, F77, G46, Q108, M173, R295, I379, I332, I111, V303, T110, Y145, Y357, I60, I63, Y437, R 144, N288, R289, H290, Q291, E302, S081, V115, K117, L113, I230, A370, H243, A378, I382, M383, T399, S81, S192, E82, E197, S56, Q85, K407, L276, K406 or Q273; or (b) a protein that has more than 70% homology with the amino acid sequence defined in (a) and has phenylalanine lyase activity.

[0031] SEQ ID NO: 1:

[0032] 。

[0033] In a preferred embodiment, in (a), the above mutation is selected from any one or more of the following mutations: N438H, C288R, C288A, C288F, C288N, C288T, C288V, Y304L, Y304C, Y304V, F90Y, P317F, P317M, S114A, S114N, S114C, M354H, V293I, C10 3E, C103S, V59L, T89V, F77D, F77I, F77S, F77Y, F77N, G46C, Q108M, M173R, R295Q, R295K , I379L, I332A, I332D, I332S, I332N, I332H, I332Q, I111S, I111T, V303E, T110G, Y145F, Y357H, I60M, I63L, I63V, Y437F, R144Y, R144V, N288V, R289I, H290L, H290I, Q291N, E30 2W, S081R, V115I, K117V, L113V, I230A, A370G, H243Y, A378Q, I382M, M383L, T399S, T399 C, S81R, S81E, S81K, S192D, E82K, E197D, S56A, Q85R, K407T, L276E, L276T, L276N, L276D, L276K, L276R, L276Y, K406L, K406M, Q273A or Q273S, where the letter before the number represents the original amino acid and the letter after the number represents the mutated amino acid.

[0034] Mutating the wild-type phenylalanine lyase with any one or more of the above-mentioned mutations can yield phenylalanine lyase mutants. These mutants, compared to the wild-type phenylalanine lyase, are better able to prepare non-natural amino acids, including but not limited to α-methyl amino acids. Furthermore, the above-mentioned reactions for preparing α-methyl amino acids can be carried out in systems including but not limited to aqueous phase systems or two-phase systems. A two-phase system refers to a system containing two physically different, separate, and coexisting phases (including but not limited to gas-liquid, liquid-liquid, gas-solid, or liquid-solid systems). A clear interface exists between these two phases, they can coexist, and each phase has relatively uniform physical and chemical properties. The liquid-liquid two-phase system includes, but is not limited to, a water-oil two-phase system or a water-insoluble organic solvent (including but not limited to organic solvents with solubility <1 g / 100 mL at 25°C).

[0035] In a preferred embodiment, the above mutation includes any one of the following amino acid mutations: N438H, N438H+C288R, N438H+Y304C, N438H+Y304V, N438H+Y304L, N438H+P317F, N438H+P317M, N438H+I379L, N438H+F90Y, N438H+C288R+S114A, N438H+C288R+S114N, N438H+C288R+M354H, N438H+C288R+V293I, N438H+C288R+C103E, N438H+C288R+V59L, N438H+C 288R+T89V, N438H+C288R+F77D, N438H+C288R+G46C, N438H+C288R+Q108M , N438H+C288R+Y304L, N438H+C288R+C103S, N438H+S114A+C288A, N438H+ S114A+C288F, N438H+S114A+C288N, N438H+S114A+C288T, N438H+S114A+C 288V, N438H+S114A+C288T+M173R, N438H+S114A+C288T+Q108M, N438H+C28 8R+S114A+M354H, N438H+C288R+S114A+C103E, N438H+C288R+S114A+T89V , N438H+C288R+Y304L+F90Y, N438H+F77Y+S114C+C288N+R295Q, N438H+77 Y+S114A+C288T+R295K, N438H+F77S+S114A+C288N+R295Q, N438H+F77S+S 114A+C288N+R295K, N438H+F77Y+S114C+C288N+R295Q+V59L, N438H+F77I+ S114C+C288N+R295Q, N438H+F77S+S114C+C288N+R295Q, N438H+F77Y+S11 4C+C288N+R295Q, N438H+F77Y+S114C+C288N+R295Q+I332D, N438H+F77Y+ S114C+C288N+R295Q+I111S, N438H+F77Y+S114C+C288N+R295Q+I111T, N4 38H+F77Y+S114C+C288N+R295Q+V303E, N438H+F77N+S114C+C288N+R295Q,N438H+F77Y+S114C+C288N+R295Q+I332S、N438H+F77Y+S114C+C288N+R295Q+T110G、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S、N438H+F77Y+S114C+C288N+R295Q+V303E+Y145F、N438H+F77Y+S114C+C288N+R295Q+V303E+I332N、N438H+F77Y+S114C+C288N+R295Q+V303E+Y357H、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I63L、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+Y437F、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I332N、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I332H、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+R144Y、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Y145F+I332N、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+I63V、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+R144V、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+N288V、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+R289I、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+H290L、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+E302W、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+I332N、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+S081R、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+K117V、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+I230A、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+A370G、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+H243Y、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+A378Q、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+I332Q、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+I382M、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T110G、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+M383L、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399S、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+S81R、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+H290I、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+S192D、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+E82K、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+E197D、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+E197D+I332A、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+E197D+S56A、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+S81E+Q85R+H243Y+I332N、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+S81K+Q85K+H243Y+I332N、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T+L276E、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T+L276T、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T+L276N, N438H+F77Y+S1 14C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T+L276D, N438H+F77Y+S114C+C288N+R295 Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T+L276K, N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+ I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T+L276Y, N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V11 5I+L113V+T399C+E302W+I332D+K407T+K406L, N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C +E302W+I332D+K407T+K406M, N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K 407T+L276R, N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T+Q273A, N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T+Q273S, C288R, Y304L or F90Y.

[0036] In this application, "+" means "and", for example, "N438H+C288R" means that the N438H mutation and the C288R mutation occur simultaneously on the basis of the wild-type phenylalanine lyase shown in SEQ ID NO: 1.

[0037] In a preferred embodiment, the phenylalanine lyase mutant comprises a protein having 80% or more, 85% or more, more preferably 95% or more, and even more preferably 99% or more homology with the amino acid sequence defined in (a) and having phenylalanine lyase activity.

[0038] All the aforementioned amino acid mutations were experimentally investigated in the embodiments of this application. Compared to the parent protein with the amino acid sequence shown in SEQ ID NO: 1, all of them exhibited the activity of catalyzing the binding of enoic acid substrates and amino donors to prepare α-methyl amino acids. All of the above mutation sites were located around the active site of the amino acid, which improves the binding ability of the mutant to the substrate and / or its catalytic activity. Mutations far from the active site have less impact on the enzyme's catalytic activity; therefore, proteins with 80% or more homology to the aforementioned amino acid sequences and the same catalytic activity can be obtained.

[0039] In this application, homology refers to the "homology" between amino acid sequences, that is, the total ratio of the same type of amino acid residues in the amino acid sequence. The homology of amino acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0040] Proteins with 70%, 75%, 80%, 85%, 90%, 95%, or more than 99% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or even more than 99.9%) homology and the same function have an active site, active pocket, active mechanism, protein structure, etc. that are highly likely to be the same as the protein provided by sequence (a), and are homologous proteins obtained through amino acid mutation.

[0041] As used herein, the amino acid residue abbreviations are as follows: alanine (Ala; A), asparagine (Asn; N), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tyrosine (Tyr; Y), and valine (Val; V).

[0042] Substitution and replacement rules generally apply to amino acids with similar properties; the effects of substituting one another are similar. For example, conserved amino acid substitutions can occur in the aforementioned homologous proteins. "Conserved amino acid substitutions" include, but are not limited to:

[0043] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0044] Hydrophobic amino acids with large side chains (Phe, Tyr) are replaced by other hydrophobic amino acids with large side chains;

[0045] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0046] Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

[0047] Those skilled in the art can also perform conservative substitutions of amino acids based on amino acid substitution rules well known to them, such as the "blosum62 score matrix" in the prior art.

[0048] The "AlphaFold2-Multimer" used in this application is a publicly available artificial intelligence model capable of predicting the conformation of protein complexes. Its predictions of protein three-dimensional structures are very close to those observed in real-world experiments using equipment such as cryo-electron microscopy. This allows for the acquisition of relatively realistic protein structures, thereby guiding the investigation of protein structure and activity.

[0049] In a second typical embodiment of this application, a DNA molecule is provided that encodes the above-mentioned phenylalanine lyase mutant.

[0050] In a third typical embodiment of this application, a recombinant plasmid is provided, which contains the aforementioned DNA molecule.

[0051] The aforementioned DNA can encode the phenylalanine lyase mutant and can be ligated onto a recombinant plasmid to form a circular DNA. Both the aforementioned DNA and the recombinant plasmid can be transcribed and translated by RNA polymerase, ribosomes, tRNA, etc., to obtain the aforementioned phenylalanine lyase mutant.

[0052] In a fourth typical embodiment of this application, a host cell is provided, which contains the aforementioned DNA molecule or the aforementioned recombinant plasmid.

[0053] In a preferred embodiment, the host cell includes Escherichia coli, optionally Escherichia coli BL21.

[0054] Using the aforementioned host cells, recombinant plasmids can be replicated within the host cells, and the DNA molecules carried on the recombinant plasmids can be transcribed and translated to obtain a large number of phenylalanine lyase mutants. Using existing technologies, phenylalanine lyase mutants can be obtained by cleaving and purifying proteins from host cells, including but not limited to *E. coli* BL21, followed by crude enzyme catalysis or other methods, and then used for subsequent catalysis of substrate nucleosides. These host cells are of non-plant origin.

[0055] In a preferred embodiment, the host cell includes a prokaryotic cell.

[0056] In a preferred embodiment, the prokaryotic cells mentioned above include Escherichia coli.

[0057] In a fifth typical embodiment of this application, a method for preparing α-methyl amino acids is provided. The method includes: using the phenylalanine lyase described above to catalyze the reaction between an olefinic acid substrate and an amino donor to prepare the α-methyl amino acid described above.

[0058] Using the above preparation method, a phenylalanine lyase mutant is used to catalyze the reaction of an olefinic acid substrate and an amino group to obtain the target product, α-methyl amino acid. The catalytic reaction routes for various olefinic acid substrates in this invention are as follows: Figure 1 As shown.

[0059] In a preferred embodiment, the above-mentioned olefinic acid substrate is The corresponding α-methyl amino acid is ; wherein R is selected from substituted or unsubstituted aryl, heteroaryl or fused ring, and the number of carbon atoms in the aryl, heteroaryl or fused ring is 4-10.

[0060] In a preferred embodiment, the fused ring comprises quinolinyl, benzimidazolyl, benzofuranyl, benzothiophenyl, isobenzofuranyl, dibenzofuranyl, dibenzothiophenyl, benzothiazolyl, benzoisothiazolyl, benzoisoxazolyl, isoquinolinyl, cenolinyl, quinazolinyl, quinoxolinyl, carbazole, phenanthroxazolyl, phenadiazolyl, benzodiazepine, or dihydroacridyl.

[0061] In a preferred embodiment, the heteroatom in the heteroaryl group is selected from one or more of nitrogen, oxygen, or sulfur.

[0062] In a preferred embodiment, the substitutions include one or more hydrogen atoms in the aryl, heteroaryl, or fused ring being independently substituted by a substituent, which includes one or more of halogen, alkyl, nitro, trifluoromethyl, phenyl, hydroxyl, or alkoxy groups.

[0063] In this application, the inventors discovered that when a phenylalanine lyase mutant reacts with an olefinic acid substrate and an amino donor, the olefinic acid substrate... R is preferably selected from substituted or unsubstituted aryl, heteroaryl, or fused rings, wherein the number of carbon atoms in the aryl, heteroaryl, or fused ring is 4-10.

[0064] In this application, the term "substituted or unsubstituted" refers to substitution by one or more substituents selected from the following: deuterium; halogen group; nitrile group; nitro group; hydroxyl group; carbonyl group; ester group; imide group; amino group; phosphine oxide group; alkoxy group; aryloxy group; alkyl thio group; aryl thio group; alkyl sulfonyl group; silyl group; boron group; alkyl group; cycloalkyl group; alkenyl group; aryl group; aralkyl group; arylenyl group; alkylaryl group; alkylamine group; aralkylamine group; heteroarylamine group; arylamine group; arylphosphine group; or heterocyclic group containing at least one of N, O, and S, or no substituent, or substitution by substituents linked together with two or more of the exemplified substituents, or no substituent. For example, the term "substituents linked together with two or more substituents" can be biphenyl. That is, biphenyl can be aryl, or can be interpreted as substituents linked together with two phenyl groups.

[0065] In this application, "alkyl" includes straight-chain alkyl and branched-chain alkyl. C1 to C20 alkyl includes, but is not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, 2-methylbutyl, n-pentyl, sec-pentyl, neopentyl, n-hexyl, neohexyl, n-heptyl, n-octyl, 2-ethylhexyl, trifluoromethyl, pentafluoroethyl, 2,2,2-trifluoroethyl, etc.

[0066] In this application, "alkoxy" refers to a group composed of the aforementioned chain alkyl group and oxygen, or a group composed of the aforementioned cycloalkyl group and oxygen, including but not limited to methoxy, ethoxy, or propoxy.

[0067] In this application, the expression Ca to Cb represents the number of carbon atoms in the group as a to b. Unless otherwise specified, this number of carbon atoms generally does not include the number of carbon atoms in the substituents. When describing C1 to C20, it includes, but is not limited to, C1, C2, C3, C4, C3, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20. Other numerical ranges are not elaborated.

[0068] In the present application, "heteroaryl" refers to an aryl having ring backbone atoms containing at least one heteroatom selected from the group consisting of N, O, and S, and may be a monocyclic ring system such as furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, thiadiazolyl, isothiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, triazinyl, tetrazinyl, triazolyl, tetrazolyl, furazanyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, etc.

[0069] In the present application, "aryl" means aryl or (sub)aryl, and the aryl refers to a monocyclic or fused polycyclic derived from an aromatic hydrocarbon, that is, it includes phenyl, biphenyl, terphenyl, naphthyl, binaphthyl, phenylnaphthyl, naphthylphenyl, fluorenyl, phenylfluorenyl, benzofluorenyl, dibenzofluorenyl, phenanthryl, phenylphenanthryl, anthryl, indenyl, terphenylene, pyrenyl, tetrabenzo[a,c,g,i]fluorene, perylenyl, chrysenyl, naphthacenyl, allene-fused fluorenyl, etc.

[0070] In the present application, "fused ring" means a structure formed by condensation with at least one benzene ring, and the fused ring includes aromatic fused rings and non-aromatic fused rings. The fused ring includes but is not limited to benzofuryl, benzothienyl, isobenzofuryl, dibenzofuryl, dibenzothienyl, benzimidazolyl, benzothiazolyl, benzisothiazolyl, benzisoxazolyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, quinoxalinyl, carbazolyl, phenoxazolyl, phenanthridinyl, benzacenaphthylenyl, dihydroacridinyl, etc.

[0071] In the present application, the "halogen" group is defined to include F, Cl, Br or I.

[0072] As used in the present application, the term "one or more" means 1 or more than 1 under reasonable conditions, for example, 2, 3, 4, 5 or 10.

[0073] Unless specified, as used herein, the attachment point of a substituent can come from any suitable position of the substituent. When the bond of a substituent is shown as passing through the bond connecting two atoms in a ring, then such a substituent can be bonded to any ring-forming atom in the ring that can be substituted.

[0074] The above atomic names in the present invention include their corresponding various isotopes. For example, hydrogen (H) includes 1 H (protium or H), 2 H (deuterium or D), etc.; carbon (C) includes 12 C, 13 C, etc.

[0075] The inventors found that by using the above-mentioned phenylalanine lyase mutant to catalyze the reaction of an enoic acid substrate with the above characteristics and an amino donor, it is possible to preferably prepare a compound having The target product, α-methyl amino acid, is well-suited for the industrial-scale production of α-methyl amino acids.

[0076] In a preferred embodiment, the above-mentioned olefinic acid substrate is selected from any one or more of the following: , , , , , , , , , , , , , , , or .

[0077] In this application, the inventors discovered that by using the above-mentioned phenylalanine lyase mutant to catalyze the reaction between an olefinic acid substrate having the above-mentioned structure and an amino donor, the target product with the chemical structure of [missing information] can be prepared more effectively. The α-methyl amino acids are well-suited for the industrial-scale production of α-methyl amino acids. In this application, the specific olefinic acid substrates and the corresponding α-methyl amino acid structures are shown in Table 1 below.

[0078] Table 1

[0079]

[0080]

[0081] In a preferred embodiment, the amino donor includes one or more of ammonium carbamate, ammonium chloride, ammonium sulfate, ammonia, or ammonium carbonate.

[0082] The above-described method utilizes a phenylalanine lyase mutant to react amino donors, including but not limited to ammonium carbamate, ammonium chloride, ammonium sulfate, ammonia, or ammonium carbonate, with various olefinic acid substrates to generate α-methyl amino acids. During this process, components such as ammonium carbamate, ammonium chloride, ammonium sulfate, ammonia, or ammonium carbonate provide a high concentration of ammonia ions to the solution used for preparing α-methyl amino acids. Furthermore, the above preparation process is carried out simultaneously in the same container and under the same reaction conditions, thus enabling one-pot preparation of α-methyl amino acids. This method exhibits high conversion rate and efficiency, significantly reducing enzyme usage and production costs, making it suitable for industrial-scale production.

[0083] In a preferred embodiment, the above reaction is carried out in an aqueous system or a two-phase system.

[0084] In a preferred embodiment, the temperature of the above reaction is 10-50°C.

[0085] In a preferred embodiment, the temperature of the above reaction is 25-45°C.

[0086] In this application, the inventors discovered that the above-described reaction for preparing α-methyl amino acids can be carried out in systems including, but not limited to, aqueous systems or two-phase systems. The conversion rate is higher when the reaction temperature for preparing α-methyl amino acids is, for example, 10-50°C (including, but not limited to, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, or 50°C), and more preferably, when the reaction temperature is, for example, 25-45°C (including, but not limited to, 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, or 45°C). Phenylalanine lyase mutants, including but not limited to whole cells, crude enzyme lysate, pure enzyme, or enzyme powder, can catalyze different enoic acids to produce the target product α-methyl amino acids. In this application, the inventors preferably use phenylalanine lyase mutants in crude enzyme lysate form in the production of α-methyl amino acids.

[0087] In a sixth typical embodiment of this application, the application of the above-described phenylalanine lyase mutant, the above-described DNA molecule, the above-described recombinant plasmid, the above-described host cell, or the above-described preparation method in the preparation of α-methyl amino acids is provided.

[0088] In a preferred embodiment, the above-mentioned α-methyl amino acid includes those having Amino acids in the structure.

[0089] In this application, the inventors discovered that the preparation methods of the above-mentioned phenylalanine lyase mutant, the above-mentioned DNA molecule, the above-mentioned recombinant plasmid, the above-mentioned host cell, or the above-mentioned α-methyl amino acid can effectively catalyze the reaction between the enoic acid substrate and the amino donor, thereby achieving the preparation of a product with... The technical advantages of the target product α-methyl amino acid are that the reaction steps are simple, the yield is high, the stereoselectivity is good, the reaction conditions are mild, the operation is simple, and it is environmentally friendly, reducing the pollution of the environment by organic solvents.

[0090] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0091] Example 1

[0092] Using wild-type phenylalanine lyase PlPAL as the parent, saturation mutations, site-directed mutations, and combinatorial mutations were performed at specific sites to construct a mutant library. The constructed mutant library was screened for high-throughput activity using 96-well plates. Mutants with high initial activity were induced in 2 L shake flasks (optimal induction conditions: 25℃, 0.1 mM IPTG overnight induction), followed by secondary screening with whole cells, crude enzyme solution, purified enzyme, or enzyme powder to obtain mutants with enhanced activity. The saturation mutant, site-directed mutant, and combinatorial mutant libraries were constructed using whole-plasmid PCR amplification. After obtaining the PCR products, they were digested with DpnI enzyme to remove the template and then transformed into *E. coli* BL21(DE3).

[0093] In this application, a high-throughput screening method is used to screen the mutant library. The specific steps are as follows:

[0094] 1. Culture of phenylalanine lyase mutant: Add 300 μL of LB medium to each well of a 96-well plate, inoculate the single clones from the agar plate into the deep-well 96-well plate, and incubate overnight at 37℃ and 200 rpm; transfer 50 μL of the overnight culture to another 96-well plate with 600 μL of LB medium added to each well, and incubate at 37℃ and 200 rpm for 3 h. When the OD600 of the culture reaches 0.6-0.8, add 0.1 mM IPTG solution and incubate overnight at 25℃ and 200 rpm for 16 h; centrifuge at 4000 rpm for 10 min, discard the supernatant, and use whole cells for the reaction. Here, whole cells refer to the bacterial sludge collected by centrifugation of the BL21(DE3) expressed phenylalanine lyase mutant.

[0095] 2. 96-well plate high-throughput screening system: Add 190 μL of 4 M ammonium carbamate solution to each well of the 96-well plate and resuspend by shaking. Then add 10 μL of 0.1 g / mL DMSO solution of 2-methyl-3-phenylacrylic acid (final concentration 5 g / mL), and react in a shaker at 30℃ and 700 rpm for 16 hours. After the reaction is complete, add 500 μL of methanol to terminate the reaction, centrifuge, collect the supernatant, and analyze by HPLC.

[0096] 3. Secondary screening of phenylalanine lyase mutants: Following the initial screening of mutants described above, mutants with enhanced activity were obtained. These were then induced in 2 L shake flasks (optimal conditions for expression induction: 25℃, 0.1 mM IPTG overnight induction). After centrifugation to obtain bacterial sludge, secondary screening was performed using whole cells, crude enzyme solution, purified enzyme, or enzyme powder of phenylalanine lyase under suitable reaction conditions. In this application, "crude enzyme solution" refers to "crude enzyme solution from wet cells." The crude enzyme solution was obtained by centrifuging to obtain bacterial sludge, adding a certain volume of ammonia solution (including but not limited to 4 M ammonium carbamate, 4 M ammonium carbonate, etc.), and then obtaining the crude enzyme solution by ultrasonic disruption of the cells.

[0097] Example 2

[0098] Using wild-type phenylalanine lyase PlPAL (abbreviated as wild type, wt) as the parent, saturation mutations were performed at specific sites, and mutants with enhanced activity were combined to construct a mutant library. The constructed mutant library was screened for high-throughput activity using 96-well plates. After induction culture in 2 L shake flasks, the catalytic activity of the mutants was detected under the following reaction conditions: 1 mL reaction system, 5 mg / mL 2-methyl-3-phenylacrylic acid (dissolved in 50 μL DMSO and added to the system), 100 mg / mL crude enzyme solution from wet cells (after centrifugation to obtain wet cell sludge, a certain volume of 4 M ammonium carbamate was added, and the cells were disrupted by sonication to obtain the crude enzyme solution from wet cells; the content of sludge in the crude enzyme solution from wet cells was 100 mg / mL), 4 M ammonium carbamate, and the reaction was carried out at 30℃ for 16 h. After the reaction, 2 mL of methanol was added to terminate the reaction, and the supernatant was collected after centrifugation, diluted 5 times with 30% methanol, and the yield was analyzed by HPLC. A schematic diagram of the chemical reaction for preparing (S)-2-2-methyl-3-phenylalanine by using a phenylalanine lyase mutant to catalyze the binding of 2-methyl-3-phenylacrylic acid and an amino donor is shown below. Figure 2 As shown.

[0099] The results of the catalytic activity detection are shown in Table 2.

[0100] Table 2

[0101]

[0102] Example 3

[0103] The optimal mutant N438H+C288R obtained in Example 2 was used as the parent for the next round of saturation mutagenesis, site-directed mutagenesis, and combinatorial mutagenesis. Mutants with improved activity were initially screened and induced in 2L shake flasks. Activity screening was then performed under the following conditions: 1 mL reaction system, 20 mg / mL 2-methyl-3-phenylacrylic acid (dissolved in 100 μL DMSO and added to the system), 100 mg / mL crude enzyme solution from wet cells (after centrifugation to obtain wet cell sludge, a certain volume of 4 M ammonium carbamate was added, and the cells were disrupted by ultrasonication to obtain the crude enzyme solution; the sludge content in the crude enzyme solution was 100 mg / mL), 4 M ammonium carbamate, and the reaction was carried out at 30℃ for 16 h. After the reaction, 2 mL of methanol was added to terminate the reaction. After centrifugation, the supernatant was collected, diluted 20-fold with 30% methanol, and the yield was analyzed by HPLC. The experimental results of activity detection are shown in Table 3.

[0104] Table 3

[0105]

[0106] Example 4

[0107] Based on the mutant N438H+F77Y+S114C+C288N+R295Q obtained in Example 3, and with a substrate addition of 20 mg, the reaction volume, enzyme amount, ammonia solution, and reaction temperature were optimized. The reaction results are shown in Table 4. In a crude enzyme solution of 500 mg / mL wet cells (after centrifugation to obtain wet cell sludge, a certain volume of 4 M ammonium carbamate was added, and the cells were disrupted by ultrasonication to obtain the crude enzyme solution of wet cells, the content of sludge in the crude enzyme solution of wet cells was 500 mg / mL), in a 4 M ammonium carbamate solution, at 30°C, the conversion rate was 41.25%. When the ammonia solution was ammonium carbonate, the conversion rate decreased to 16.62%. At different temperatures, the conversion rate was 37.82% at 50°C.

[0108] Table 4

[0109]

[0110] Example 5

[0111] Using the mutant N438H+F77Y+S114C+C288N+R295Q obtained in Example 3 as the parent, a second round of saturation mutagenesis, site-directed mutagenesis, and combinatorial mutagenesis was performed. Mutants with improved activity were initially screened and induced in 2L shake flasks. Activity screening was then conducted under the following reaction conditions: 1 mL reaction system, 20 mg / mL 2-methyl-3-phenylacrylic acid (dissolved in 100 μL DDMSO and added to the system), 60 mg / mL crude enzyme solution from wet cells (after centrifugation to obtain wet cell sludge, a certain volume of 4M ammonium carbamate was added, and the cells were disrupted by ultrasonication to obtain the crude enzyme solution from wet cells; the sludge content in the crude enzyme solution was 60 mg / mL), 4M ammonium carbamate, and the reaction was carried out at 50℃ for 16 h. After the reaction, 2 mL of methanol was added to terminate the reaction. After centrifugation, the supernatant was collected, diluted 20-fold with 30% methanol, and the yield was analyzed by HPLC. The experimental results are shown in Table 5.

[0112] Table 5

[0113]

[0114] Example 6

[0115] The phenylalanine lyase mutant N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N, obtained by saturation mutagenesis, site-directed mutagenesis, and combinatorial mutagenesis of wild-type phenylalanine lyase in Example 5, was used as the parent for the next round of saturation mutagenesis, site-directed mutagenesis, and combinatorial mutagenesis. The mutants with improved activity after initial screening were induced and cultured in 2 L shake flasks, and activity screening was carried out according to the following reaction conditions: 1 mL reaction system, 20 mg / mL 2-methyl-3-phenylacrylic acid (dissolved in 100 μL DMSO and added to the system), 20 mg / mL crude enzyme solution of wet cells (after centrifugation to obtain wet cell sludge, a certain volume of 4 M ammonium carbamate was added, and the crude enzyme solution of wet cells was obtained by ultrasonic disruption of cells, the content of sludge in the crude enzyme solution of wet cells was 20 mg / mL), 4 M ammonium carbamate, and reaction at 50℃ for 16 h. After the reaction was completed, 2 mL of methanol was added to the system to terminate the reaction. After centrifugation, the supernatant was collected, diluted 20 times with 30% methanol, and the yield was analyzed by HPLC. The experimental results are shown in Table 6.

[0116] Table 6

[0117]

[0118]

[0119] Example 7

[0120] Add 800 mL of 4 M ammonium carbamate solution to a 5 L reactor, then add 20 g of 2-methyl-3-phenylacrylic acid (dissolved in 100 mL of DMSO before being added to the system). Heat the system to 50 °C, then add 100 mL of a solution containing 20 g of DMSO. Crude enzyme solution of wet cells from mutant strain N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T+Q273S (after centrifuging 50g of bacterial sludge from the N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T+Q273S mutant strain, a certain volume of 4 M ammonium carbonate solution was added, and the crude enzyme solution of wet cells was obtained by ultrasonic cell disruption. The total volume of this crude enzyme solution was 150mL). 64 The reaction was completed in h. 3 L of methanol was added to the system to denature the enzyme, filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to 200 mL. The pH was adjusted to about 7.0 with hydrochloric acid, 1 L of methanol was added, and the mixture was placed at low temperature for 16 h before filtration to obtain 17.93 g of the target product (S)-2-methyl-3-phenylalanine.

[0121] HPLC analysis showed a purity >98%, L configuration, ee value (Enanatiomeric Excess, which indicates the excess of one enantiomer over another; a higher ee value indicates higher optical purity of the corresponding enantiomer) >99%, and a yield of 79.5%.

[0122] Example 8

[0123] The optimal mutant obtained was validated for substrate applicability. A 1 mL reaction system was prepared with 10 mg / mL of different types of olefinic acid substrates (dissolved in 50 μL DMSO before being added to the system), 50 mg of enzyme powder, and 4 M ammonium carbamate. The reaction was carried out at 40 °C for 64 h. After the reaction was completed, 2 mL of methanol was added to terminate the reaction. The supernatant was collected after centrifugation, diluted 5-fold with 30% methanol, and the yield was analyzed by HPLC.

[0124] The best conversion rates for different substrates are shown in Table 7. Substrate 1 has the following structure: The structure of substrate 2 is The structure of substrate 3 is The structure of substrate 4 is The structure of substrate 5 is as follows: The structure of substrate 6 is The structure of substrate 7 is The structure of substrate 8 is The structure of substrate 9 is The structure of substrate 10 is as follows: The structure of substrate 11 is as follows: The structure of substrate 12 is as follows: The structure of substrate 13 is as follows: The structure of substrate 14 is as follows: The structure of substrate 15 is as follows: The structure of substrate 16 is The structure of substrate 17 is The conversion rates of different substrates are shown in Table 7.

[0125] Table 7

[0126]

[0127] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects:

[0128] In this application, a series of phenylalanine lyase mutants were obtained through phenylalanine lyase evolution. The results showed that these mutants exhibited significantly enhanced catalytic activity compared to wild-type phenylalanine lyase. By utilizing these phenylalanine lyase mutants, the synthesis of the target product α-methyl amino acid was achieved through the catalytic combination of different types of enoic acid substrates and amino donors. The reaction steps are simple, with high yield, good stereoselectivity, mild reaction conditions, and simple operation, making it environmentally friendly and reducing pollution from organic solvents. The phenylalanine lyase synthesis method boasts 100% atom economy and excellent stereoselectivity, advantages that are difficult to match with traditional chemical methods. It highly aligns with the urgent needs of the modern pharmaceutical industry for green, efficient, and safe production, and offers significant advantages such as mild reaction conditions, excellent stereoselectivity, and environmental friendliness.

[0129] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A phenylalanine lyase mutant, characterized in that, include: (a) A protein mutated based on the wild-type phenylalanine lyase shown in SEQ ID NO: 1, wherein the mutation is selected from mutations at any one or more of the following sites: N438, C288, Y304, F90, P317, S114, M354, V293, C103, V59, T89, F77, G46, Q108, M173, R295, I379, I332, I111, V303, T110, Y14 5. Y357, I60, I63, Y437, R144, N288, R289, H290, Q291, E302, S081, V115, K117, L113, I230, A370, H243, A378, I382, M383, T399, S81, S192, E82, E197, S56, Q85, K407, L276, K406 or Q273; or (b) A protein that has more than 70% homology with the amino acid sequence defined in (a) and has phenylalanine lyase activity.

2. The phenylalanine lyase mutant according to claim 1, characterized in that, In (a), the mutation is selected from any one or more of the following mutations: N438H, C288R, C288A, C288F, C288N, C288T, C288V, Y304L, Y304C, Y304V, F90Y, P31 7F, P317M, S114A, S114N, S114C, M354H, V293I, C103E, C103S, V59L, T89V, F77D, F7 7I, F77S, F77Y, F77N, G46C, Q108M, M173R, R295Q, R295K, I379L, I332A, I332D, I33 2S, I332N, I332H, I332Q, I111S, I111T, V303E, T110G, Y145F, Y357H, I60M, I63L, I 63V, Y437F, R144Y, R144V, N288V, R289I, H290L, H290I, Q291N, E302W, S081R, V115 I, K117V, L113V, I230A, A370G, H243Y, A378Q, I382M, M383L, T399S, T399C, S81R, S 81E, S81K, S192D, E82K, E197D, S56A, Q85R, K407T, L276E, L276T, L276N, L276D, L276K, L276R, L276Y, K406L, K406M, Q273A or Q273S, where the letter before the number represents the original amino acid and the letter after the number represents the mutated amino acid.

3. The phenylalanine lyase mutant according to claim 1, characterized in that, The mutation includes any one of the following amino acid mutations: N438H、N438H+C288R、N438H+Y304C、N438H+Y304V、N438H+Y304L、N438H+P317F、N438H+P317M、N438H+I379L、N438H+F90Y、N438H+C288R+S114A、N438H+C288R+S114N、N438H+C288R+M354H、N438H+C288R+V293I、N438H+C288R+C103E、N438H+C288R+V59L、N438H+C288R+T89V、N438H+C288R+F77D、N438H+C288R+G46C、N438H+C288R+Q108M、N438H+C288R+Y304L、N438H+C288R+C103S、N438H+S114A+C288A、N438H+S114A+C288F、N438H+S114A+C288N、N438H+S114A+C288T、N438H+S114A+C288V、N438H+S114A+C288T+M173R、N438H+S114A+C288T+Q108M、N438H+C288R+S114A+M354H、N438H+C288R+S114A+C103E、N438H+C288R+S114A+T89V、N438H+C288R+Y304L+F90Y、N438H+F77Y+S114C+C288N+R295Q、N438H+77Y+S114A+C288T+R295K、N438H+F77S+S114A+C288N+R295Q、N438H+F77S+S114A+C288N+R295K、N438H+F77Y+S114C+C288N+R295Q+V59L、N438H+F77I+S114C+C288N+R295Q、N438H+F77S+S114C+C288N+R295Q、N438H+F77Y+S114C+C288N+R295Q、N438H+F77Y+S114C+C288N+R295Q+I332D、N438H+F77Y+S114C+C288N+R295Q+I111S、N438H+F77Y+S114C+C288N+R295Q+I111T、N438H+F77Y+S114C+C288N+R295Q+V303E、N438H+F77N+S114C+C288N+R295Q、N438H+F77Y+S114C+C288N+R295Q+I332S、N438H+F77Y+S114C+C288N+R295Q+T110G、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S、N438H+F77Y+S114C+C288N+R295Q+V303E+Y145F、N438H+F77Y+S114C+C288N+R295Q+V303E+I332N、N438H+F77Y+S114C+C288N+R295Q+V303E+Y357H、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I63L、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+Y437F、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I332N、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I332H、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+R144Y、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Y145F+I332N、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+I63V、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+R144V、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+N288V、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+R289I、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+H290L、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+E302W、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+I332N、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+S081R、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+K117V、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+I230A、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+A370G、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+H243Y、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+A378Q、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+I332Q、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+I382M、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T110G、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+M383L、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399S、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+S81R、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+H290I、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+S192D、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+E82K、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+E197D、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+E197D+I332A、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+E197D+S56A、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+S81E+Q85R+H243Y+I332N、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+S81K+Q85K+H243Y+I332N、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T+L276E、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T+L276T、N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T+L276N, N438H+F77Y+S1 14C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T+L276D, N438H+F77Y+S114C+C288N+R295 Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T+L276K, N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+ I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T+L276Y, N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V11 5I+L113V+T399C+E302W+I332D+K407T+K406L, N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C +E302W+I332D+K407T+K406M, N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K 407T+L276R, N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T+Q273A, N438H+F77Y+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T+Q273S, C288R, Y304L or F90Y.

4. A DNA molecule, characterized in that, The DNA molecule encodes the phenylalanine lyase mutant according to any one of claims 1 to 3.

5. A recombinant plasmid, characterized in that, The recombinant plasmid contains the DNA molecule as described in claim 4.

6. A host cell, characterized in that, The host cell contains the DNA molecule of claim 4 or the recombinant plasmid of claim 5.

7. A method for preparing α-methyl amino acids, characterized in that, The preparation method includes: using the phenylalanine lyase according to any one of claims 1 to 3 to catalyze the reaction of an olefinic acid substrate and an amino donor to prepare the α-methyl amino acid.

8. The preparation method according to claim 7, characterized in that, The olefinic acid substrate is The corresponding α-methyl amino acid is Wherein, R is selected from substituted or unsubstituted aryl, heteroaryl, or fused rings, and the number of carbon atoms in the aryl, heteroaryl, or fused ring is 4-10.

9. The preparation method according to claim 8, characterized in that, The fused ring includes quinolinyl, benzimidazolyl, benzofuranyl, benzothiophenyl, isobenzofuranyl, dibenzofuranyl, dibenzothiophenyl, benzothiazolyl, benzoisothiazolyl, benzoisooxazolyl, isoquinolinyl, cenolinyl, quinazolinyl, quinoxolinyl, carbazole, phenanthroxazolyl, phenidyl, benzodiazepine, or dihydroacridyl.

10. The preparation method according to claim 8, characterized in that, The heteroatom in the heteroaryl group is selected from one or more of nitrogen, oxygen, or sulfur.

11. The preparation method according to claim 8, characterized in that, The substitution comprises one or more hydrogen atoms in the aryl, heteroaryl, or fused ring being independently substituted by a substituent, which includes one or more of halogen, alkyl, nitro, trifluoromethyl, phenyl, hydroxy, or alkoxy groups.

12. The preparation method according to claim 7, characterized in that, The olefinic acid substrate is selected from any one or more of the following: , , , , , , , , , , , , , , , or .

13. The use of the phenylalanine lyase mutant of any one of claims 1 to 3, the DNA molecule of claim 4, the recombinant plasmid of claim 5, the host cell of claim 6, or the preparation method of any one of claims 7 to 12 in the preparation of α-methyl amino acids.

14. The application according to claim 13, characterized in that, The α-methyl amino acid includes those having Amino acids in the structure.

Images