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CN-122012481-A - Phenylalanine lyase mutant and preparation method of amino acid

CN122012481ACN 122012481 ACN122012481 ACN 122012481ACN-122012481-A

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 based on the wild-type phenylalanine lyase shown in SEQ ID NO. 1, wherein the mutation is selected from any one or more of F90, S107, P317, C288, F77, R307, M529, H290, S114, C103 and the like, or (b) a protein having 70% or more homology with the amino acid sequence defined in (a) and having phenylalanine lyase activity. The phenylalanine lyase mutant can be used for realizing the efficient synthesis of amino acid, especially unnatural amino acid, and is suitable for the field of enzyme catalysis.

Inventors

  • ZHANG NA
  • JIAO XUECHENG
  • ZHANG KEJIAN
  • HUANG AIPING
  • YANG YIMING
  • LI JUAN
  • SU LANKAI
  • YOU TIAN
  • ZHAO XIN

Assignees

  • 天津凯莱英生物科技有限公司
  • 凯莱英医药集团（天津）股份有限公司

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 mutation ：F90、S107、P317、C288、F77、R307、M529、H290、S114、C103、Q108、W119、G46、K79、V293、R295、I379、I380、I381、H447、I332、V303 or Q413 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: 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、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, wherein the letter before the number represents the original amino acid and the letter after the number represents the mutated amino acid.
  3. 3. The phenylalanine lyase mutant according to claim 1, wherein the mutation comprises 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. 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 amino acid, which comprises catalyzing the reaction between an olefinic acid substrate and an amino donor by 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 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 preparation method according to any one of claims 7-12 for the preparation of an amino acid.
  14. 14. The use according to claim 13, wherein the amino acid comprises a polypeptide having Amino acids of the structure.

Description

Phenylalanine lyase mutant and preparation method of 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 amino acid. Background Amino acids, which are indispensable small molecular organic compounds in living bodies, are not only basic stones for protein construction, but also deeply participate in a wide range of physiological processes, such as regulation of enzyme activity, regulation of metabolic pathways, signal transmission, construction of cell structures, and the like. Amino acids include natural amino acids and unnatural amino acids, wherein unnatural amino acids have wide application prospects in the fields of biological medicine, basic research, material science, synthetic biology and the like due to their 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 chemical method for synthesizing the unnatural amino acid mainly comprises a natural amino acid derivatization method and an asymmetric synthesis method. However, the natural amino acid derivatization method is limited to the original structure, and it is difficult to synthesize unnatural amino acids with large structural differences. In addition, asymmetric synthesis methods are generally cumbersome in steps, involving not only protection and deprotection of amino and carboxyl groups, but also the use of heavy metal catalysts or expensive chiral ligands. Therefore, developing a simple and efficient synthetic method for unnatural amino acids has become a key scientific problem for associating basic research with industrial applications, and has great market demands. Disclosure of Invention The main purpose of the invention is to provide a phenylalanine lyase mutant and a preparation method of amino acid, and the phenylalanine lyase mutant can be used for realizing high-efficiency synthesis of amino acid, especially 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 ：F90、S107、P317、C288、F77、R307、M529、H290、S114、C103、Q108、W119、G46、K79、V293、R295、I379、I380、I381、H447、I332、V303 or Q413 at any one or more of the following sites, or (b) a protein having a homology of 70% or more 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 ：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、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, 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 ：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. In order to achieve the above object, according to a second aspect of the present invention, there is provided a DNA molecule encoding the phenylalanine lyase mutant described above. In order to achieve the above object, according to a third aspect of the present invention, there is provided a recombinant plasmid containing the above DNA molecule. In order to achieve the above object, according to a fourth aspect of the present invention, there is pro