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CN-122012482-A - Phenylalanine lyase mutant and preparation method of alpha-methyl amino acid

CN122012482ACN 122012482 ACN122012482 ACN 122012482ACN-122012482-A

Abstract

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

Inventors

  • ZHANG NA
  • JIAO XUECHENG
  • ZHANG KEJIAN
  • HUANG AIPING
  • SU LANKAI
  • YOU TIAN
  • ZHAO XIN

Assignees

  • 天津凯莱英生物科技有限公司
  • 天津凯莱英制药有限公司

Dates

Publication Date
20260512
Application Date
20260210

Claims (14)

  1. 1. A phenylalanine lyase mutant, which has the functions of improving immunity, characterized by comprising the following steps: (a) A protein mutated based on the wild-type phenylalanine lyase shown in SEQ ID NO. 1, said mutation being selected from the group consisting of a mutation ：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、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 at any one or more of the following positions, or (B) A protein having a phenylalanine lyase activity and having a homology of 70% or more with the amino acid sequence defined in (a).
  2. 2. The phenylalanine lyase mutant according to claim 1, wherein in (a) the mutation is selected from any one or more of the following: 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、I379L、I332A、I332D、I332S、I332N、I332H、I332Q、I111S、I111T、V303E、T110G、Y145F、Y357H、I60M、I63L、I63V、Y437F、R144Y、R144V、N288V、R289I、H290L、H290I、Q291N、E302W、S081R、V115I、K117V、L113V、I230A、A370G、H243Y、A378Q、I382M、M383L、T399S、T399C、S81R、S81E、S81K、S192D、E82K、E197D、S56A、Q85R、K407T、L276E、L276T、L276N、L276D、L276K、L276R、L276Y、K406L、K406M、Q273A Or Q273S, wherein the pre-number letter represents the original amino acid and the post-number letter represents the mutant amino acid.
  3. 3. The phenylalanine lyase mutant according to claim 1, wherein the mutation comprises 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+S114C+C288N+R295Q+V303E+I111S+I60M+Q291N+V115I+L113V+T399C+E302W+I332D+K407T+L276D、N438H+F77Y+S114C+C288N+R295Q+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+V115I+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+K407T+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. 4. A DNA molecule encoding the phenylalanine lyase mutant according to any one of claims 1-3.
  5. 5. A recombinant plasmid is characterized in that, the recombinant plasmid contains the DNA molecule of claim 4.
  6. 6. A host cell comprising the DNA molecule of claim 4 or the recombinant plasmid of claim 5.
  7. 7. A process for producing an alpha-methylamino acid, which comprises preparing the alpha-methylamino acid by catalyzing a reaction between an enoate substrate and an amino donor using the phenylalanine lyase according to any one of claims 1 to 3.
  8. 8. The method of claim 7, wherein the enoate substrate is The corresponding alpha-methyl amino acid is Wherein R is selected from a substituted or unsubstituted aryl, heteroaryl or fused ring, and the number of carbon atoms in the aryl, heteroaryl or fused ring is 4-10.
  9. 9. The method according to claim 8, wherein, the condensed ring comprises quinolinyl, benzimidazolyl, benzofuranyl, benzothienyl, isobenzofuranyl, and dibenzofuranyl, dibenzothiophenyl, benzothiazolyl, benzisothiazolyl, benzofuranyl, dibenzothiophenyl, benzisothiazolyl, benzidine, benzi dibenzofuranyl, dibenzothienyl, dibenzoyl benzothiazolyl, benzisothiazolyl, benzothiazolyl, benzisothiazolyl, benzidine-containing compounds, benzi.
  10. 10. The method of claim 8, wherein the heteroatom in the heteroaryl is selected from one or more of nitrogen, oxygen, or sulfur.
  11. 11. The method of preparation of claim 8, wherein the substitution comprises one or more hydrogen atoms in the aryl, heteroaryl, or fused ring each independently substituted with a substituent comprising one or more of halogen, alkyl, nitro, trifluoromethyl, phenyl, hydroxy, or alkoxy.
  12. 12. The method of claim 7, wherein the enoate substrate is selected from any one or more of the following: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 Or (b) 。
  13. 13. Use of the phenylalanine lyase mutant according to any one of claims 1-3, the DNA molecule according to claim 4, the recombinant plasmid according to claim 5, the host cell according to claim 6, or the method of preparation according to any one of claims 7-12 for the preparation of an alpha-methyl amino acid.
  14. 14. The use according to claim 13, wherein the alpha-methyl amino acid comprises a polypeptide having Amino acids of the structure.

Description

Phenylalanine lyase mutant and preparation method of alpha-methyl amino acid Technical Field The invention relates to the field of enzyme catalysis, in particular to a phenylalanine lyase mutant and a preparation method of alpha-methyl amino acid. Background The unnatural amino acid has wide application prospect in the fields of biological medicine, basic research, material science, synthetic biology and the like due to the unique physical, chemical and biological properties. As a key component of many bioactive molecules and drugs, unnatural amino acids are effective in enhancing the stability of peptide chains and proteins and in enhancing their resistance to chemical and enzymatic degradation. The existing method for synthesizing the unnatural amino acid is mainly a chemical synthesis method, however, the chemical synthesis method has the problems of low yield of the unnatural amino acid, use of toxic reagent sodium cyanide, complicated process for preparing chiral precursors, air sensitivity of an organic metal catalyst, high cost of chiral ligands and the like. Therefore, the method for synthesizing the unnatural amino acid, which has the advantages of low cost and easy availability of raw materials, simple reaction system and easy operation, has become a key scientific problem for related basic research and industrial application and has huge market demands. Disclosure of Invention The invention mainly aims to provide a phenylalanine lyase mutant and a preparation method of alpha-methyl amino acid, which are used for solving the problem that the prior art is difficult to simply and efficiently synthesize unnatural amino acid. In order to achieve the above object, according to a first aspect of the present invention, there is provided a phenylalanine lyase mutant comprising (a) a protein mutated based on a wild-type phenylalanine lyase represented by SEQ ID NO. 1, the mutation being selected from the group consisting of mutation ：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、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 at any one or more of the following sites, or (b) a protein having 70% or more homology with the amino acid sequence defined in (a) and having phenylalanine lyase activity. Further, in the above (a), the mutation is selected from any one or more of mutation ：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、I379L、I332A、I332D、I332S、I332N、I332H、I332Q、I111S、I111T、V303E、T110G、Y145F、Y357H、I60M、I63L、I63V、Y437F、R144Y、R144V、N288V、R289I、H290L、H290I、Q291N、E302W、S081R、V115I、K117V、L113V、I230A、A370G、H243Y、A378Q、I382M、M383L、T399S、T399C、S81R、S81E、S81K、S192D、E82K、E197D、S56A、Q85R、K407T、L276E、L276T、L276N、L276D、L276K、L276R、L276Y、K406L、K406M、Q273A or Q273S, wherein the letter before the number represents the original amino acid and the letter after the number represents the mutated amino acid. Further, 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+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+I6