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CN-122012477-A - Phenylalanine aminomutase and application thereof in preparation of R-type beta-amino acid

CN122012477ACN 122012477 ACN122012477 ACN 122012477ACN-122012477-A

Abstract

The invention discloses phenylalanine ammonia mutase and application thereof in preparation of R-type beta-amino acid. The phenylalanine ammonia mutase has an amino acid sequence comprising one or more amino acid residue differences in positions 59, 60, 144, 233, 247, 281, 304, 330, 394, 396, 399, 403 and 427 compared to the amino acid sequence shown in SEQ ID NO. 1. The invention adopts the enzyme to synthesize the (R) -beta-phenylalanine derivative, which can obviously improve the regioselectivity, the enantioselectivity and the conversion rate of the product.

Inventors

  • DONG HE
  • LI JIE
  • CHANG YAOWEN
  • HUANG JIAO

Assignees

  • 常州合全药业有限公司

Dates

Publication Date
20260512
Application Date
20241108

Claims (10)

  1. 1. A phenylalanine ammonia mutase is characterized in that the amino acid sequence of the phenylalanine ammonia mutase is different from the amino acid sequence shown in SEQ ID NO. 1 by comprising one or more amino acid residues at positions 59, 60, 144, 233, 247, 281, 304, 330, 394, 396, 399, 403 and 427.
  2. 2. The phenylalanine ammonia mutase according to claim 1 wherein the amino acid sequence of the phenylalanine ammonia mutase comprises one or more of the following amino acid residue differences compared to the amino acid sequence shown in SEQ ID No. 1: (1) E59L, L144V, P233C, Q247L, L A, L394R, C G/Q/P/R, E399D, Y F/L/V/T and/or N427G; (2) L60G/S/N, G281C, L F and/or L330M; Preferably, the amino acid sequence of the phenylalanine ammonia mutase comprises one or more of the following amino acid residue differences E399D, preferably one or more selected from the group consisting of E59L, L G/S/N, L144V, P233C, G281C, L304F, L M and C396R, compared to the amino acid sequence shown in SEQ ID NO:1, preferably the amino acid sequence of the phenylalanine ammonia mutase comprises one of the following combinations of amino acid residue differences compared to the amino acid sequence shown in SEQ ID NO: 1: (1) E399D and L144V; (2) E399D, L V and C396R; (3) E399D, L V and E59L; (4) E399D, G281C and E59L; (5) E399D and G281C; Further preferably, the amino acid sequence of phenylalanine ammonia mutase comprises the combination of amino acid residue differences E399D, L V and C396R, and further comprises one or more selected from the group consisting of L60G/S/N and L330M, as compared to the amino acid sequence shown in SEQ ID NO. 1; more preferably, the amino acid sequence of phenylalanine ammonia mutase comprises a combination of amino acid residues of E399D, L144V, C396R and L60N, or E399D, L144V, C396R and L60S, compared to the amino acid sequence shown in SEQ ID NO. 1.
  3. 3. 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 mutase according to claim 1 or 2, and (2) (1) The complement of the polynucleotide sequence; Preferably, the polynucleotide sequence of (1) is shown in SEQ ID NO. 6, 7 or 8, preferably in SEQ ID NO. 8.
  4. 4. A nucleic acid construct comprising the nucleic acid molecule according to claim 3, wherein the nucleic acid construct is an expression cassette.
  5. 5. A recombinant vector comprising the nucleic acid molecule of claim 3 or the nucleic acid construct of claim 4; preferably, the recombinant vector is a recombinant cloning vector or a recombinant expression vector; more preferably, the backbone of the recombinant vector is pET-30a.
  6. 6. A transformant comprising the nucleic acid molecule of claim 3, the nucleic acid construct of claim 4 or the recombinant vector of claim 5, and/or expressing the phenylalanine ammonia mutase of claim 1 or 2; 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 E.coli, e.g.BL 21 (DE 3).
  7. 7. An enzyme preparation comprising the phenylalanine ammonia mutase according to claim 1 or 2.
  8. 8. Use of a phenylalanine ammonia mutase according to claim 1 or 2, a nucleic acid molecule according to claim 3, a nucleic acid construct according to claim 4, a recombinant vector according to claim 5, a transformant according to claim 6 or an enzyme preparation according to claim 7 for the preparation of a compound according to formula I; Wherein R is hydrogen, halogen or hydroxy; preferably, the compound shown in the formula I is (R) -3-amino-3-phenylpropionic acid or (R) -3-amino-3-p-chlorophenyl propionic acid; More preferably, when preparing the compound of formula I, trans-cinnamic acid or p-chlorocinnamic acid is used as a substrate.
  9. 9. 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 in a formula II by using the phenylalanine ammonia mutase shown in claim 1 or 2 or the enzyme preparation shown in claim 7 to prepare the compound shown in the formula I; Wherein R is hydrogen, halogen or hydroxy; Preferably, the compound shown in the formula I is (R) -3-amino-3-phenylpropionic acid or (R) -3-amino-3-p-chlorophenyl propionic acid, and the compound shown in the formula II is trans-cinnamic acid or p-chlorocinnamic acid; Further preferably, the amino donor is an ammonium carbonate buffer; More preferably, the mass of phenylalanine ammonia mutase is 3.7-11.1 times the weight of the compound of formula I.
  10. 10. The method of claim 9, wherein the reaction conditions of the method are selected from one or more of the following: (1) The pH of the reaction is 6.0-10.0, preferably 8.0-10.0, more preferably 9.5; (2) The reaction temperature is 10-50 ℃, preferably 20-45 ℃, more preferably 30 ℃; (3) The reaction time is 0.1 to 48 hours, preferably 0.5 to 24 hours, more preferably 10 to 24 hours; (4) The concentration of the ammonium carbonate buffer is 2-6mol/L, for example 4mol/L.

Description

Phenylalanine aminomutase and application thereof in preparation of R-type beta-amino acid Technical Field The invention belongs to the technical field of biochemistry, and particularly relates to phenylalanine ammonia mutase and application thereof in preparing R-type beta-amino acid. Background Ammonia lyase (EC 4.3.1. X) has the function of reversibly catalyzing C-N bond cleavage reactions, catalyzing the formation of alpha-and beta-amino acid backbones into ammonia and the corresponding unsaturated or cyclic derivatives (Turner N J.Curr Opin Chem biol.15:234-240[2011 ]). Phenylalanine ammonia mutase (PAL), along with Histidine Ammonia Lyase (HAL) and Tyrosine Ammonia Lyase (TAL), are members of the aromatic amino acid lyase family (EC 4.3.1.23-1.25 and 4.3.1.3). 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 ]). 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). However, enzymes of natural origin are often more active under milder natural conditions, but less active under reaction conditions of more extreme industrial application, in particular when the catalytic substrate is also a non-natural substrate. The engineered PAL variants are reported to be active on cinnamic acid having a small substituent on the aromatic ring (see, e.g., gloge et al chem. Eur. J.,6:3386-3390[2000 ]). The PAL used in the present invention is derived from the genus chondrocyte hooked (Chondromyces crocatus) and the substrate used is trans-cinnamic acid, the present invention being optimized to provide enhanced catalytic activity in order to further realize the industrial application of the enzyme in the reaction. Disclosure of Invention In order to solve the technical problems in the prior art, the invention provides phenylalanine ammonia mutase and application thereof in preparing R-type beta-amino acid. The technical problems are solved by the following technical scheme. In a first aspect the invention provides a phenylalanine ammonia mutase whose amino acid sequence differs from the amino acid sequence shown in SEQ ID NO. 1 by one or more amino acid residues comprised in positions 59, 60, 144, 233, 247, 281, 304, 330, 394, 396, 399, 403 and 427. In some embodiments of the invention, the amino acid sequence of the phenylalanine ammonia mutase comprises one or more of the following amino acid residue differences compared to the amino acid sequence shown in SEQ ID NO. 1: (1) E59L, L144V, P233C, Q247L, L A, L394R, C G/Q/P/R, E399D, Y F/L/V/T and/or N427G; (2) L60G/S/N, G281C, L F and/or L330M. Wherein "/" in "C396G/Q/P/R", "Y403F/L/V/T" and "L60G/S/N" means that the relationship between the amino acid residues which are mutable at the same site is "OR", and the same applies to the similar descriptions at other sites below. In some embodiments of the invention, the amino acid sequence of the phenylalanine ammonia mutase comprises an amino acid residue difference of E399D compared to the amino acid sequence shown in SEQ ID NO. 1, preferably further comprises one or more selected from the group consisting of E59L, L G/S/N, L144V, P233C, G281C, L304F, L M and C396R, preferably the amino acid sequence of the phenylalanine ammonia mutase comprises one of the following combinations of amino acid residue differences compared to the amino acid sequence shown in SEQ ID NO. 1: (1) E399D and L144V; (2) E399D, L V and C396R; (3) E399D, L V and E59L; (4) E399D, G281C and E59L; (5) E399D and G281C. In some embodiments of the invention, the amino acid sequence of phenylalanine ammonia mutase comprises a combination of amino acid residue differences of E399D, L V and C396R, and further comprises one or more amino acid residue differences selected from the group consisting of L60G/S/N and L330M, as compared to the amino acid sequence shown in SEQ ID NO. 1. In some embodiments of the invention, the amino acid sequence of phenylalanine ammonia mutase comprises a combination of amino acid residue differences as compared to the amino acid sequence shown in SEQ ID NO.1, E399D, L144V, C396R and L60N, or E399D, L144V, C396R and L60S. In a second aspect the invention provides an isolated nucleic acid molecule having a sequence selected from the group consisting of: (1) Polynucleotide sequence encoding a phenylalanine am