Preparation method of phenylalanine lyase mutant and amino acid

Abstract

The invention provides a phenylalanine lyase mutant and a preparation method of 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: F90, S107, P317, C288, F77, R307, M529, H290, S114, C103 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 phenylalanine lyase mutant can realize efficient synthesis of amino acids, especially non-natural amino acids, 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 phenylalanine lyase mutant and a method for preparing amino acids. Background Technology

[0002] Amino acids, as essential small organic molecules in living organisms, are not only the building blocks of proteins but also deeply involved in a wide range of physiological processes, such as the regulation of enzyme activity, the control of metabolic pathways, signal transduction, and the construction of cell structures. Amino acids are broadly classified into natural and non-natural amino acids. Non-natural amino acids, due to their unique physical, chemical, and biological properties, show great promise for applications in 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 chemical methods for synthesizing non-natural amino acids include natural amino acid derivatization and asymmetric synthesis. However, natural amino acid derivatization is limited by the existing structure, making it difficult to synthesize non-natural amino acids with significant structural differences. Furthermore, asymmetric synthesis methods are typically cumbersome, involving not only the protection and deprotection of amino and carboxyl groups but also the use of heavy metal catalysts or expensive chiral ligands. Therefore, developing simple and efficient methods for synthesizing non-natural amino acids 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 phenylalanine lyase mutant and a method for preparing amino acids, which enables the efficient synthesis of amino acids, especially non-natural amino acids, using this phenylalanine lyase mutant.

[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: F90, S107, P317, C288, F77, R307, M529, H290, S114, C103, Q108, W119, G46, K79, V293, R295, I379, I380, I381, H447, I332, V303, or Q413; or (b) a protein having more than 70% homology with the amino acid sequence defined in (a) and having phenylalanine lyase activity.

[0006] Further, in (a) above, the mutation is selected from any one or more of the following mutations: F90L, F90V, F90E, F90T, F90Q, S107T, P317Q, P317G, P317W, P317L, P317F, C288A, C288N, C288M, C288D, C288V, C288E, C288R, C288K, C288L, C288H, C288I, F77L, F77A, R307G, R307R, R307K, M529S The following amino acids are listed: M529K, M529E, H290T, S114T, C103G, C103Q, Q108M, Q108L, W119F, G46V, K79G, V293H, V293E, V293F, R295S, R295N, R295G, R295D, R295Q, I379L, I380L, I381L, H447T, I332N, V303N, Q413C, or Q413S. The letters preceding the numbers represent the original amino acids, and the letters following the numbers represent the mutated amino acids.

[0007] Further, the above mutations include any one of the following amino acid mutations: F90L, F90L+M529S, F90L+M529K, F90L+M529E, F90L+H290T, F90L+C288A, F90L+C288N, F90L+C288M, F90F+C288D, F90L+C288L, F90C+C288V, F90L+C288E, F90L+C288R, F90L+C288H, F90L+C288I, F90L+P317W, F90L+P317Q, F90L+S114T, F90L+R307R, F90L+S107T, F90L+C103G, F9 0L+Q108M, F90L+W119F, F90L+F77L, F90L+F77A, F90L+G46V, F90L+S114T, F 90L+K79G, F90L+Q108L, F90L+V293H, F90L+V293E, F90L+R295S, F90L+R295N , F90L+R295G, F90L+R295D, F90L+I379L, F90L+I380L, F90L+I381L, F90L+C 288E+C103Q, F90L+C288E+P317Q, F90L+C288E+R295Q, F90L+C288E+V293F, F 90L+C288E+G46V, F90L+C288K+H447T, F90L+C288N+R295S, F90L+C288R+R2 95N, F90L+C288R+R295G, F90L+C288R+R295D, F90L+C288R+I332N, F90L+C28 8R+V303N, F90L+C288R+Q108M, F90L+C288R+S114T, F90L+C288R+C103G, F9 0L+C288R+R307G, F90L+C288R+R307K, F90L+C288R+P317L, F90L+C288R+P31 7F, F90L+C288R+V293H, F90L+C288R+V293E, F90L+C288R+I379L, F90L+C28 8R+Q413C, F90L+C288R+Q413S, F90V, F90E, F90T, F90Q, S107T, P317Q, P317G , P317W, P317L, P317F, C288A, C288N, C288M, C288D, C288V, C288E, C288R, C 288K, C288L, C288H, C288I, F77L, F77A, R307G, R307R, R307K, M529S, M529K,M529E, H290T, S114T, C103G, C103Q, Q108M, Q108L, W119F, G46V, K79G, V293H, V293E, V293F, R295S, R295N, R295G, R295D, R295Q, I379L, I380L, I381L, H447T, I332N, V303N, Q413C or Q413S.

[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 objectives, according to a fifth aspect of the present invention, a method for preparing an amino acid 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 amino acid.

[0013] Furthermore, the above-mentioned olefinic acid substrate is The corresponding amino acids mentioned above are ; 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 ring includes quinolinyl, benzimidazolyl, benzofuranyl, benzothiopheneyl, isobenzofuranyl, dibenzofuranyl, dibenzothiopheneyl, benzothiazolyl, benzoisothiazolyl, benzoisoxazolyl, isoquinolinyl, cenolinyl, quinazolinyl, quinoxolinyl, carbazole, phenanthroxazolyl, phenadiazolyl, benzodiazepine, or 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 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 are provided for use in the preparation of amino acids.

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

[0024] By applying the technical solution of this invention, the aforementioned phenylalanine lyase mutant can efficiently catalyze the binding of different enoic acid substrates with amino acid donors to prepare amino acids. Compared to the wild-type enzyme, the aforementioned phenylalanine lyase mutant exhibits higher conversion rates and efficiency in amino acid preparation, highly meeting the urgent needs of the 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 The catalytic production of 3-(quinoline-3-yl)acrylic acid according to Example 2 of the present invention is shown. S A schematic diagram of the chemical reaction of 3-(quinolin-3-yl)alanine. 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 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 of 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: F90, S107, P317, C288, F77, R307, M529, H290, S114, C103, Q108, W119, G46, K79, V293, R295, I379, I380, I381, H447, I332, V303, or Q413; or (b) a protein having more than 70% homology with the amino acid sequence defined in (a) and having phenylalanine lyase activity.

[0031] In a preferred embodiment, in (a), the above mutation is selected from any one or more of the following mutations: F90L, F90V, F90E, F90T, F90Q, S107T, P317Q, P317G, P317W, P317L, P317F, C288A, C288N, C288M, C288D, C288V, C288E, C288R, C288K, C288L, C288H, C288I, F77L, F77A, R307G, R307R, R307K, M52 9S, M529K, M529E, H290T, S114T, C103G, C103Q, Q108M, Q108L, W119F, G46V, K79G, V293H, V293E, V293F, R295S, R295N, R295G, R295D, R295Q, I379L, I380L, I381L, H447T, I332N, V303N, Q413C or Q413S, where the letter before the number represents the original amino acid and the letter after the number represents the mutated amino acid.

[0032] 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, can better prepare, but are not limited to, the target amino acid product. Furthermore, the above-mentioned amino acid preparation reaction can be carried out in, but is not limited to, aqueous 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).

[0033] In a preferred embodiment, the above mutation includes any one of the following amino acid mutations: F90L, F90L+M529S, F90L+M529K, F90L+M529E, F90L+H290T, F90L+C288A, F90L+C288N, F90L+C288M, F90F+C288D, F90L+C288L, F90C+C288V, F90L+C288E, F90L+C288R, F90L+C288H, F90L+C288I, F90L+P317W, F90L+P317Q, F90L+S114T, F90L+R307R, F90L+S107T, F90L+C1 03G, F90L+Q108M, F90L+W119F, F90L+F77L, F90L+F77A, F90L+G46V, F90L+S 114T, F90L+K79G, F90L+Q108L, F90L+V293H, F90L+V293E, F90L+R295S, F90L +R295N, F90L+R295G, F90L+R295D, F90L+I379L, F90L+I380L, F90L+I381L, F90L+C288E+C103Q, F90L+C288E+P317Q, F90L+C288E+R295Q, F90L+C288E+V 293F, F90L+C288E+G46V, F90L+C288K+H447T, F90L+C288N+R295S, F90L+C2 88R+R295N, F90L+C288R+R295G, F90L+C288R+R295D, F90L+C288R+I332N, F9 0L+C288R+V303N, F90L+C288R+Q108M, F90L+C288R+S114T, F90L+C288R+C1 03G, F90L+C288R+R307G, F90L+C288R+R307K, F90L+C288R+P317L, F90L+C28 8R+P317F, F90L+C288R+V293H, F90L+C288R+V293E, F90L+C288R+I379L, F9 0L+C288R+Q413C, F90L+C288R+Q413S, F90V, F90E, F90T, F90Q, S107T, P317Q , P317G, P317W, P317L, P317F, C288A, C288N, C288M, C288D, C288V, C288E, C 288R, C288K, C288L, C288H, C288I, F77L, F77A, R307G, R307R, R307K, M529S,M529K, M529E, H290T, S114T, C103G, C103Q, Q108M, Q108L, W119F, G46V, K79G, V293H, V293E, V293F, R295S, R295N, R295G, R295D, R295Q, I379L, I380L, I381L, H447T, I332N, V303N, Q413C or Q413S.

[0034] SEQ ID NO: 1:

[0035] .

[0036] In this application, "+" means "and", for example, "F90L+C288R" means that the F90L 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 mutations exhibited the activity of catalyzing the binding of enoic acid substrates and amino donors to prepare amino acids. All the above mutation sites were located around the active site of the amino acid, which improves the binding ability and / or catalytic activity of the mutant to the substrate. Mutations far from the active site have less impact on the enzyme's catalytic activity, thus enabling the production of proteins with 80% or more homology to the aforementioned amino acid sequences and identical catalytic activity.

[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 in this article, the amino acid residue abbreviations are as follows: arginine (Arg; R), cysteine ​​(Cys; C), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), tryptophan (Trp; W), 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 (Trp, Cys, Gly, Pro, Met, Val, Ile) are replaced by other hydrophobic amino acids;

[0044] The hydrophobic amino acid with a large side chain (Phe) is 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, 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 an amino acid is provided, the method comprising: using the phenylalanine lyase described above to catalyze the reaction of an olefinic acid substrate and an amino donor to prepare the 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 acid donor to prepare the target product, an 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 amino acids mentioned above are ; 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 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 this application, "heteroaryl" refers to an aryl group containing a ring backbone atom selected from the group consisting of N, O, and S, and can be a monocyclic ring such as furanyl, thiophene, pyrrole, imidazolyl, pyrazolyl, thiazolyl, thiadiazolyl, isothiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, triazinyl, tetraazinyl, triazolyl, tetraazolyl, furazolidyl, pyridinyl, 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 aromatic hydrocarbons, including, for example, phenyl, biphenyl, terphenyl, naphthyl, binaphthyl, phenylnaphthyl, naphthylphenyl, fluorenyl, phenylfluorenyl, benzofluorenyl, dibenzofluorenyl, phenanthryl, phenylphenanthryl, anthryl, indenyl, terphenylene, pyrenyl, tetrabenzo[ghi]perylenyl, perylenyl, pleiadienyl, 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, including but not limited to benzofuranyl, benzothiophenyl, isobenzofuranyl, dibenzofuranyl, dibenzothiophenyl, 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, such as 2, 3, 4, 5 or 10.

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

[0074] The atomic names described above in the present invention include their corresponding various isotopes. For example, hydrogen (H) includes 1 H (protium or also called H), 2 H (deuterium or also called 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, an amino acid with the target product chemical structure of can be preferably prepared, which is preferably applicable to the industrial scale production of amino acids.

[0076] In a preferred embodiment, the above enoic 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 amino acids are well-suited for the industrial-scale production of amino acids. In this application, the specific olefinic acid substrates and their corresponding 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 acid donors, including but not limited to ammonium carbamate, ammonium chloride, ammonium sulfate, ammonia, or ammonium carbonate, with various olefinic acid substrates to generate 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 amino acid preparation solution. 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 amino acids. This method exhibits high conversion rate and efficiency, significantly reducing enzyme usage and consequently lowering 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-mentioned amino acid preparation reaction can be carried out in a system including but not limited to an aqueous phase or a two-phase system. The conversion rate is higher when the reaction temperature is, for example, 10-50℃ (including but not limited to 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, or 50℃), and more preferably, when the reaction temperature is, for example, 25-45℃ (including but not limited to 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, or 45℃). Phenylalanine lyase mutants, including but not limited to whole cells, crude enzyme lysate, pure enzyme, or enzyme powder, can effectively catalyze different enoic acids to produce the target product, amino acids. In this application, the inventors preferably use phenylalanine lyase mutants in the crude enzyme lysate state in the amino acid production process.

[0087] In a sixth typical embodiment of this application, the application 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 preparation method in the preparation of amino acids is provided.

[0088] In a preferred embodiment, the above-mentioned amino acids include 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 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 effects of amino acid structures.

[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), followed by secondary screening with whole cells, crude enzyme solution, 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. Mutant Culture: Add 300 μL of LB medium to each well of a 96-well plate. Inoculate the single clones from the agar plates into the deep-well 96-well plates and incubate overnight at 37°C and 200 rpm. Transfer 50 μL of the overnight culture to another 96-well plate with 600 μL of LB medium per well. Incubate at 37°C and 200 rpm for 3 hours. When the OD600 of the culture reaches 0.6-0.8, add 0.1 mM IPTG solution and incubate overnight at 25°C and 200 rpm for about 16 hours. 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 3-(quinoline-3-yl)acrylic 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 mutants: Following the initial screening, 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 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 using ultrasonic cell disruption to obtain the crude enzyme solution.

[0097] Example 2

[0098] wild-type phenylalanine lyase Pl Using PAL (wt) as the parent culture, saturation mutations were performed at specific sites to construct a mutant library. The constructed mutant library was then subjected to high-throughput activity screening using 96-well plates. Mutants with high initial activity were induced and cultured in 2 L shake flasks. The catalytic activity of the mutants was then tested under the following reaction conditions: 1 mL reaction system, 10 mg / mL 3-(quinoline-3-yl)acrylic 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 ultrasonication to obtain the crude enzyme solution from wet cells; 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 5 times with 30% methanol, and the yield was analyzed by HPLC. Using a phenylalanine lyase mutant, the reaction of 3-(quinoline-3-yl)acrylic acid and carbamate was catalyzed to prepare ( S A schematic diagram of the chemical reaction of 3-(quinolin-3-yl)alanine is shown below. Figure 2 As shown.

[0099] The test results are shown in Table 2.

[0100] Table 2

[0101]

[0102] Example 3

[0103] The optimal mutant F90L obtained in Example 1 was further optimized in terms of reaction volume, enzyme amount, ammonia solution, and reaction temperature with the addition of 50 mg of substrate 3-(quinoline-3-yl)acrylic acid. The reaction results are shown in Table 3. In a crude enzyme solution of 50 mg / mL wet cells (after centrifugation to obtain bacterial 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, with bacterial sludge content of 50 mg / mL), and in a 4 M ammonium carbamate solution, at 30 °C, the conversion rate was 64.76%. When the ammonia solution was ammonium carbonate, the conversion rate increased to 65.62%. At different temperatures, the conversion rate was 72.36% at 40 °C.

[0104] Table 3

[0105]

[0106] Example 4

[0107] Using the optimal mutant F90L obtained in Example 1 as the parent, the next round of saturation mutagenesis, site-directed mutagenesis, and combinatorial mutagenesis was conducted. Mutants with improved activity were initially screened and induced in 2L shake flasks. Activity screening was then performed under the following reaction conditions: 1 mL reaction system, 50 mg / mL 3-(quinoline-3-yl)acrylic acid (dissolved in 100 μL DMSO and added to the system), 50 mg / mL crude enzyme solution from wet cells (after centrifugation to obtain bacterial sludge, a certain volume of 4 M ammonium carbonate solution was added, and the cells were disrupted by sonication to obtain the crude enzyme solution from wet cells; the bacterial sludge content in the crude enzyme solution from wet cells was 50 mg / mL), 4 M ammonium carbonate solution, and the reaction was carried out at 40℃ 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 results are shown in Table 4.

[0108] Table 4

[0109]

[0110] Example 5

[0111] 850 mL of 4 M ammonium carbonate solution was added to a 5 L reactor, followed by 50 g of 3-(quinoline-3-yl)acrylic acid (dissolved in 100 mL of DMSO before being added to the system). The system was heated to 40 °C, and 150 mL of crude enzyme solution containing 50 g of F90L+C288M wet cells was added (after centrifuging to obtain 50 g of bacterial sludge from the F90L+C288M mutant strain, a certain volume of 4 M ammonium carbonate solution was added, and the cells were disrupted by ultrasonication to obtain the crude enzyme solution; the total volume of this crude enzyme solution was 150 mL). The reaction was allowed to proceed for 16 h until complete. 3 L of methanol was added to the system to denature the enzyme, and the mixture was filtered through diatomaceous earth. The filtrate was concentrated under reduced pressure to 200 mL, and the pH was adjusted to approximately 7.0 with hydrochloric acid. 1 L of methanol was added, and the mixture was allowed to stand at low temperature for 16 h before filtration to obtain 45.66 g of the target product.

[0112] HPLC analysis showed a purity >98%, L configuration, ee value (Enanatiomeric Excess, which indicates the excess of one enantiomer over another; a higher value indicates higher optical purity of the corresponding enantiomer) >99%, and a yield of 89.5%.

[0113] Example 6

[0114] We used what we obtained PlPAL mutants were used to validate the activity of different substrates. A 1 mL reaction system was prepared, containing 10 mg / mL of different types of olefinic acid substrates (dissolved in 50 μL DMSO before being added to the system), 10 mg of enzyme powder, and 4 M ammonium carbamate. The reaction was carried out at 30°C for 16 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. The structure of substrate 1 was [not specified]. 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 The structure of substrate 13 is as follows: The structure of substrate 14 is The structure of substrate 15 is The structure of substrate 16 is The structure of substrate 17 is The conversion rates of different substrates are shown in Table 5.

[0115] Table 5

[0116]

[0117] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects:

[0118] In this application, a series of phenylalanine lyase mutants were obtained through phenylalanine lyase evolution. The results showed that these mutants achieved high yields and selectivity for various types of olefinic acid substrates. The reaction steps are simple, with high yields, good stereoselectivity, mild reaction conditions, and easy operation. It possesses the ability to produce the target amino acid product, is environmentally friendly, and reduces 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 has significant advantages such as mild reaction conditions, excellent stereoselectivity, and environmental friendliness.

[0119] 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: F90, S107, P317, C288, F77, R307, M529, H290, S114, C103, Q108, W119, G46, K79, V293, R295, I379, I380, I381, H447, I332, V303, or Q413; 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: F90L, F90V, F90E, F90T, F90Q, S107T, P317Q, P317G, P317W, P317L, P317F, C288A, C288N, C288M, C288D, C2 88V, C288E, C288R, C288K, C288L, C288H, C288I, F77L, F77A, R307G, R307R, R307K, M529S, M529K, M529E, H2 90T, S114T, C103G, C103Q, Q108M, Q108L, W119F, G46V, K79G, V293H, V293E, V293F, R295S, R295N, R295G, R295D, R295Q, I379L, I380L, I381L, H447T, I332N, V303N, Q413C or Q413S, 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: F90L、F90L+M529S、F90L+M529K、F90L+M529E、F90L+H290T、F90L+C288A、F90L+C288N、F90L+C288M、F90F+C288D、F90L+C288L、F90C+C288V、F90L+C288E、F90L+C288R、F90L+C288H、F90L+C288I、F90L+P317W、F90L+P317Q、F90L+S114T、F90L+R307R、F90L+S107T、F90L+C103G、F90L+Q108M、F90L+W119F、F90L+F77L、F90L+F77A、F90L+G46V、F90L+S114T、F90L+K79G、F90L+Q108L、F90L+V293H、F90L+V293E、F90L+R295S、F90L+R295N、F90L+R295G、F90L+R295D、F90L+I379L、F90L+I380L、F90L+I381L、F90L+C288E+C103Q、F90L+C288E+P317Q、F90L+C288E+R295Q、F90L+C288E+V293F、F90L+C288E+G46V、F90L+C288K+H447T、F90L+C288N+R295S、F90L+C288R+R295N、F90L+C288R+R295G、F90L+C288R+R295D、F90L+C288R+I332N、F90L+C288R+V303N、F90L+C288R+Q108M、F90L+C288R+S114T、F90L+C288R+C103G、F90L+C288R+R307G、F90L+C288R+R307K、F90L+C288R+P317L、F90L+C288R+P317F、F90L+C288R+V293H、F90L+C288R+V293E、F90L+C288R+I379L、F90L+C288R+Q413C、F90L+C288R+Q413S、F90V、F90E、F90T、F90Q、S107T、P317Q、P317G、P317W、P317L、P317F、C288A、C288N、C288M、C288D、C288V、C288E、C288R、C288K、C288L、C288H、C288I、F77L、F77A、R307G、R307R、R307K、M529S、M529K、M529E、H290T、S114T、C103G、C103Q, Q108M, Q108L, W119F, G46V, K79G, V293H, V293E, V293F, R295S, R295N, R295G, R295D, R295Q, I379L, I380L, I381L, H447T, I332N, V303N, Q413C or Q413S.

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 an amino acid, 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 amino acid.

8. The preparation method according to claim 7, characterized in that, The olefinic acid substrate is The corresponding 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 amino acids.

14. The application according to claim 13, characterized in that, The amino acids include those having Amino acids in the structure.

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