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CN-121991937-A - Phenylalanine ammonia lyase and application thereof in preparation of S-type beta-amino acid

CN121991937ACN 121991937 ACN121991937 ACN 121991937ACN-121991937-A

Abstract

The invention discloses phenylalanine ammonia lyase and application thereof in preparation of S-type beta-amino acid. The amino acid sequence of the phenylalanine ammonia lyase differs from the amino acid sequence shown in SEQ ID NO. 1 by one or more amino acid residues at positions 147, 244, 251, 314, 333 and 342. The invention adopts the mutant of phenylalanine ammonia lyase from streptomyces and takes cinnamic acid or derivatives thereof as a substrate to prepare S-type beta-amino acid, and the regioselectivity, the enantioselectivity and the conversion rate of the product are all obviously improved.

Inventors

  • SHAO QI
  • LI JIE
  • HUANG JIAO
  • CHANG YAOWEN

Assignees

  • 常州合全药业有限公司

Dates

Publication Date
20260508
Application Date
20241108

Claims (11)

  1. 1. A phenylalanine ammonia lyase characterized in that the amino acid sequence of the phenylalanine ammonia lyase differs from the amino acid sequence shown in SEQ ID NO.1 by one or more amino acid residues at positions 147, 244, 251, 314, 333 and 342.
  2. 2. The phenylalanine ammonia lyase according to claim 1 wherein the phenylalanine ammonia lyase comprises one or more amino acid residue differences compared to the amino acid sequence shown in SEQ ID NO.1 selected from the group consisting of L147T, V244L, R251T, R I/S/M, L333I/P and N342S; Preferably, the phenylalanine ammonia lyase comprises a combination of amino acid residue differences L147T and V244L compared to the amino acid sequence as shown in SEQ ID NO. 1, preferably further comprising one or more of the amino acid residue differences R251T, R I/S M, L333I/P and N342S; further preferably, the phenylalanine ammonia lyase comprises the following combination of amino acid residue differences compared to the amino acid sequence as shown in SEQ ID NO.1, L147T, V L and R314I/S/M; More preferably, L147T, V L and R314I are included together with one or more amino acid residue differences selected from the group consisting of L333I/P and N342S, or R251T is included together with L147T, V244L, R M; even more preferably, the phenylalanine ammonia lyase comprises a combination of amino acid residue differences L147T, V244L, R I and N342S, preferably also amino acid residue differences L333I/P, compared to the amino acid sequence as shown in SEQ ID NO. 1.
  3. 3. The phenylalanine ammonia-lyase according to claim 1 wherein the phenylalanine ammonia-lyase comprises one selected from the group consisting of the following combinations of amino acid residue differences compared to the amino acid sequence shown in SEQ ID No. 1: (1) L147T and V244L; (2) L147T, V244L and R314I; (3) L147T, V244L and R314S; (4) L147T, V244L, R M and R251T; (5) L147T, V244L, R I and N342S; (6) L147T, V244L, R I, L333P and N342S; (7) L147T, V244L, R314I, L333I and N342S.
  4. 4. An isolated nucleic acid molecule, wherein the sequence of the nucleic acid molecule is selected from the group consisting of: (1) A polynucleotide sequence encoding the phenylalanine ammonia lyase according to any one of claims 1-3, and (2) The complement of the polynucleotide sequence of (1); preferably, the polynucleotide sequence of (1) is shown in SEQ ID NO. 5 or 6, preferably in SEQ ID NO. 6.
  5. 5. A nucleic acid construct comprising the nucleic acid molecule of claim 4, wherein the nucleic acid construct is an expression cassette.
  6. 6. A recombinant vector comprising the nucleic acid molecule of claim 4 or the nucleic acid construct of claim 5; preferably, the recombinant vector is a recombinant cloning vector or a recombinant expression vector.
  7. 7. A transformant comprising the nucleic acid molecule according to claim 4, the nucleic acid construct according to claim 5 or the recombinant vector according to claim 6 and/or expressing the phenylalanine ammonia lyase according to any one of claims 1-3; preferably, the host cell used in the construction of the transformant is selected from the group consisting of E.coli cells, insect cells, yeast cells and mammalian cells; more preferably, the host cell is an E.coli cell, such as BL21 (DE 3).
  8. 8. An enzyme preparation comprising the phenylalanine ammonia lyase according to any one of claims 1-3; preferably, the enzyme preparation is a liquid preparation or a freeze-dried powder; Further preferably, the liquid preparation comprises a buffer selected from the group consisting of phosphate buffer, ammonium carbonate buffer and triethanolamine-isopropylamine buffer, wherein the pH is preferably 6-11, more preferably 7-10, and even more preferably 9-10; More preferably, the buffer solution is an ammonium carbonate buffer solution, and the concentration of the buffer solution is preferably 1-10M, and more preferably 2-6M.
  9. 9. Use of the phenylalanine ammonia lyase according to any one of claims 1-3, the nucleic acid molecule according to claim 4, the nucleic acid construct according to claim 5, the recombinant vector according to claim 6, the transformant according to claim 7 or the enzyme preparation according to claim 8 for the preparation of a compound according to formula I; Wherein R is hydrogen, methyl, ethyl or halogen; Preferably, the compound shown in the formula I is (S) -beta-phenylalanine or chlorine- (S) -beta-phenylalanine; more preferably, cinnamic acid or p-chlorocinnamic acid is used as a substrate in the preparation of the compound shown in the formula I.
  10. 10. A process for preparing a compound of formula I, comprising the steps of: Catalyzing an amino donor to contact and react with a compound shown as a formula II by using the phenylalanine ammonia lyase according to any one of claims 1-3 or the enzyme preparation according to claim 8 to prepare the compound shown as the formula I; Wherein R is hydrogen, methyl, ethyl or halogen; Preferably, the compound shown in the formula I is (S) -beta-phenylalanine, the compound shown in the formula II is cinnamic acid, or the compound shown in the formula I is chlorine- (S) -beta-phenylalanine, the compound shown in the formula II is p-chlorocinnamic acid, and/or the amino donor is ammonium carbonate buffer solution; more preferably, the phenylalanine ammonia lyase is 1-50% by mass, preferably 10-40% by mass, more preferably 15-30% by mass of the compound represented by formula II.
  11. 11. The method of claim 10, wherein the method comprises reacting the phenylalanine ammonia lyase with the compound of formula II in the presence of a co-solvent to produce the compound of formula I; preferably, the reaction conditions of the process are selected from one or more of the following: (1) The concentration of the ammonium carbonate buffer solution is preferably 1-10M, more preferably 2-6M; (2) The pH value of the ammonium carbonate buffer solution is 6-11, preferably 7-10, more preferably 9-10, and even more preferably 9-9.5; (3) The reaction temperature is 10-65 ℃, preferably 40-60 ℃, more preferably 50-55 ℃; (4) The reaction time is 0.1 to 120 hours, preferably 0.5 to 48 hours, more preferably 10 to 24 hours; (5) The cosolvent is dimethyl sulfoxide, an alcohol solvent, toluene or a combination thereof, preferably dimethyl sulfoxide.

Description

Phenylalanine ammonia lyase and application thereof in preparation of S-type beta-amino acid Technical Field The invention belongs to the technical field of biochemistry, and particularly relates to phenylalanine ammonia lyase and application thereof in preparation of S-type beta-amino acid. Background Amino acid derivatives are important intermediates, and can be widely used in the fields of pharmaceutical synthesis, chemical industry and the like, wherein unnatural amino acid derivatives are difficult to industrially produce because of relatively difficult synthesis and relatively expensive cost (Walker, K.D.biol chem.279,53947-53954[2004 ]). Ammonia lyase (EC 4.3.1. X) has the function of reversibly catalyzing C-N bond cleavage reactions, in particular for alpha-amino acids, to form ammonia and the corresponding unsaturated or cyclic derivatives (Turner N J.Curr Opin Chem biol.15:234-240[2011 ]). Phenylalanine ammonia lyase (PAL, EC 4.3.1.24/25), histidine ammonia lyase (HAL, EC 4.3.1.3) and tyrosine ammonia lyase (TAL, EC 4.3.1.23/25) belong to the family of aromatic amino acid lyases in ammonia lyases (Fabio P.chem.Rev.118:73-118[2018 ]). The family of lyase is a structurally and mechanically similar enzyme that catalyzes the deamination of aromatic amino acids to the corresponding aryl acrylic acid in nature. Accordingly, it is also possible to use its reverse reaction to catalyze the ammonification of aryl acrylic acid to aromatic amino acids, in which process ammonia can be introduced either in the alpha position or in the beta position (Turner, N.J. chem biol 15,234-240[2011 ]). As with other ammonia lyase, the PAL protein structure near the active site also contains a cofactor unit consisting of Ala-Ser-Gly with an electrophilic capacity, 3, 5-dihydro-5-methylene-4H-imidazol-4-one (MIO) (Alina F.ChemCatchem.10:2627-2633[2018 ]). MIO units may attack the aromatic ring of the substrate in Friedel-Craft (FC) mode, thereby causing electron rearrangement and C-N bond cleavage (Sarolta P.chem. Eur.18:7793-7802[2012 ]). The current activity of ammonia cleavage of PAL and the formation of the corresponding unsaturated or cyclic derivative may allow its use in the preparation of compounds with partly medicinal potential. Furthermore, PAL is reversibly reduced to form unsaturated or cyclic derivatives under high ammonia concentration conditions, which results in highly specific selective amination products, which makes PAL also useful for Chiral preparation or Chiral resolution of the corresponding substrate (AR Aguillon. Biological toward THE SYNTHESIS of Chiral amino acids John Wiley & Sons, ltd 2020). Most of the natural enzymes are active better under mild conditions and are less active under industrially applicable reaction conditions, with worse results if they catalyze substrates other than natural substrates. Disclosure of Invention In order to solve the technical problems in the prior art, the invention provides phenylalanine ammonia lyase and application thereof in preparing S-type beta-amino acid. The mutant obtained by optimizing the wild type enzyme PAL from the streptomyces (Streptomyces maritimus) uses cinnamic acid or derivatives thereof instead of L-phenylalanine as a natural substrate, and the catalytic regioselectivity, enantioselectivity and activity of the mutant are improved obviously. The technical problems are solved by the following technical scheme. In a first aspect the invention provides a phenylalanine ammonia lyase having an amino acid sequence comprising one or more of the amino acid residue differences 147, 244, 251, 314, 333 and 342 compared to the amino acid sequence shown in SEQ ID NO. 1. In some embodiments of the invention, the phenylalanine ammonia lyase comprises one or more amino acid residue differences from the amino acid sequence shown in SEQ ID NO. 1 selected from the group consisting of L147T, V244L, R251T, R314I/S/M, L333I/P and N342S. Wherein "/" in "R314I/S/M" means that the relationship between amino acid residues which are mutable at the same site is "OR", and the same is true for the similar descriptions at other sites below. In some embodiments of the invention, the phenylalanine ammonia lyase comprises a combination of amino acid residue differences L147T and V244L compared to the amino acid sequence shown in SEQ ID NO.1, e.g., the amino acid sequence of the phenylalanine ammonia lyase is shown in SEQ ID NO. 2. In some embodiments of the invention, the phenylalanine ammonia lyase comprises a combination of amino acid residue differences L147T and V244L as compared to the amino acid sequence shown in SEQ ID NO. 1, and further comprises one or more amino acid residue differences selected from the group consisting of R251T, R314I/S/M, L333I/P and N342S. In some embodiments of the invention, the phenylalanine ammonia lyase comprises a combination of amino acid residue differences L147T, V L and R314I/S/M compared to the amino acid sequence shown as SEQ ID NO.