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

Abstract

The invention discloses phenylalanine ammonia lyase and application of the phenylalanine ammonia lyase in preparation of S-type beta-amino acid. Compared with an amino acid sequence as shown in SEQ ID NO: 1, the amino acid sequence of the phenylalanine ammonia lyase comprises one or more amino acid residue differences in the following sites: the 147th site, the 244th site, the 251st site, the 314th site, the 333th site and the 342nd site. The S-type beta-amino acid is prepared by adopting a mutant of phenylalanine ammonia lyase from streptomyces and taking cinnamic acid or a derivative thereof as a substrate, and the regioselectivity, the enantiomer selectivity and the conversion rate of the product are all remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically relating to a phenylalanine aminolyase and its application in the preparation of S-type β-amino acids. Background Technology

[0002] Amino acid derivatives are an important intermediate that can be widely used in drug synthesis and chemical engineering. However, non-natural amino acid derivatives are difficult to synthesize and are expensive, making industrial production difficult (Walker, KDBiol. Chem. 279, 53947-53954

[2004] ).

[0003] Aminolysin (EC 4.3.1.X) has the function of reversibly catalyzing the cleavage of CN bonds, especially for α-amino acids, to generate ammonia and corresponding unsaturated or cyclic derivatives (Turner N J. Curr Opin Chem Biol. 15:234-240

[2011] ). Phenylalanine aminolysin (PAL, EC 4.3.1.24 / 25), histidine aminolysin (HAL, EC 4.3.1.3), and tyrosine aminolysin (TAL, EC 4.3.1.23 / 25) belong to the aromatic amino acid lyase family of aminolysins (Fabio P. Chem. Rev. 118:73-118

[2018] ). This family of lyases is a structurally and mechanistically similar enzyme that, in nature, catalyzes the deamination of aromatic amino acids to generate the corresponding arylacrylic acid. Correspondingly, it can also use its reverse reaction to catalyze the addition of ammonia to arylacrylic acid to generate aromatic amino acids. In this process, ammonia can be introduced into both the α- and β-positions (Turner, NJ Chem. Biol. 15, 234-240

[2011] ).

[0004] Like other amino acid lyases, the PAL protein also contains an electrophilic cofactor unit composed of Ala-Ser-Gly near its active site, 3,5-dihydro-5-methylene-4H-imidazol-4-one (MIO) (Alina F. Chem. Cat. Chem. 10: 2627-2633

[2018] ). The MIO unit may attack the aromatic ring of the substrate via the Friedel-Craft (FC) mode, thereby causing electronic rearrangement and CN bond cleavage (Sarolta P. Chem. Eur. 18: 7793-7802

[2012] ).

[0005] The current activity of PAL in ammonia cleavage to generate corresponding unsaturated or cyclic derivatives allows it to be used in the preparation of some compounds with pharmaceutical potential. Furthermore, under high concentrations of ammonia, PAL can also reversibly reduce the unsaturated or cyclic derivatives generated by amination to generate highly specific and selective amination products. This characteristic allows PAL to also be used for the chiral preparation or chiral resolution of corresponding substrates (AR Aguillon. Biocatalysis toward the Synthesis of Chiral Amines. John Wiley & Sons, Ltd.

[2020] ).

[0006] Most natural enzymes exhibit better activity under mild conditions, but poorer activity under industrial reaction conditions, and the results are even worse if the substrate they catalyze is not a natural substrate. Summary of the Invention

[0007] To address the aforementioned technical problems in the prior art, this invention provides a phenylalanine amino acid lyase and its application in the preparation of S-type β-amino acids. This invention utilizes a mutant of the wild-type enzyme PAL from the genus *Streptomyces maritimus*, optimized with cinnamic acid or its derivatives as the substrate, rather than its natural substrate L-phenylalanine. This mutant exhibits significantly improved regioselectivity, enantioselectivity, and activity.

[0008] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0009] A first aspect of the present invention provides a phenylalanine aminohydrolase whose amino acid sequence differs from that shown in SEQ ID NO:1 by one or more amino acid residues at the following sites: position 147, position 244, position 251, position 314, position 333, and position 342.

[0010] In some embodiments of the present invention, the phenylalanine aminotransferase, compared to the amino acid sequence shown in SEQ ID NO:1, comprises one or more amino acid residues selected from the following differences: L147T, V244L, R251T, R314I / S / M, L333I / P, and N342S. The " / " in "R314I / S / M" indicates an "OR" relationship between mutable amino acid residues at the same site, and similar descriptions are given for the other sites below.

[0011] In some embodiments of the present invention, the phenylalanine aminolysin contains the following amino acid residue differences compared to the amino acid sequence shown in SEQ ID NO:1: L147T and V244L. For example, the amino acid sequence of the phenylalanine aminolysin is shown in SEQ ID NO:2.

[0012] In some embodiments of the present invention, the phenylalanine aminotransferase, compared with the amino acid sequence shown in SEQ ID NO:1, comprises the following amino acid residue differences: L147T and V244L, and further comprises one or more amino acid residue differences selected from the following: R251T, R314I / S / M, L333I / P, and N342S.

[0013] In some embodiments of the present invention, the phenylalanine amino lyase contains the following amino acid residue differences compared to the amino acid sequence shown in SEQ ID NO:1: L147T, V244L, and R314I / S / M.

[0014] In some embodiments of the present invention, the phenylalanine aminotransferase, compared with the amino acid sequence shown in SEQ ID NO:1, includes L147T, V244L, and R314I, and further includes one or more of the following amino acid residue differences: L333I / P and N342S; or, in addition to L147T, V244L, and R314M, it also includes R251T.

[0015] In some embodiments of the present invention, the phenylalanine aminolysin contains the following amino acid residue differences compared to the amino acid sequence shown in SEQ ID NO:1: L147T, V244L, R314I, and N342S, and preferably also includes the following amino acid residue difference: L333I / P; when it preferably also includes the following amino acid residue difference: L333I, the amino acid sequence of the phenylalanine aminolysin is, for example, as shown in SEQ ID NO:3.

[0016] In some specific embodiments of the present invention, the phenylalanine aminolysase, compared with the amino acid sequence shown in SEQ ID NO:1, contains the following combinations of amino acid residues with different characteristics: L147T, V244L, R314I, L333I, and N342S.

[0017] In some embodiments of the present invention, the phenylalanine aminolysase, compared with the amino acid sequence shown in SEQ ID NO:1, comprises one of the following combinations of amino acid residue differences:

[0018] (1) L147T and V244L;

[0019] (2) L147T, V244L and R314I;

[0020] (3) L147T, V244L and R314S;

[0021] (4) L147T, V244L, R314M and R251T;

[0022] (5) L147T, V244L, R314I and N342S;

[0023] (6) L147T, V244L, R314I, L333P and N342S;

[0024] (7) L147T, V244L, R314I, L333I and N342S.

[0025] A second aspect of the present invention provides an isolated nucleic acid molecule, the sequence of which is selected from:

[0026] (1) A polynucleotide sequence encoding phenylalanine aminolysin as described in the first aspect of the present invention; and

[0027] The complementary sequence of the polynucleotide sequence described in (2)(1).

[0028] In some embodiments of the present invention, (1) the polynucleotide sequence is as shown in SEQ ID NO:5 or 6, preferably as shown in SEQ ID NO:6.

[0029] A third aspect of the present invention provides a nucleic acid construct containing a nucleic acid molecule as described in the second aspect of the present invention; the nucleic acid construct is an expression cassette. The expression cassette further includes elements such as a promoter and a terminator.

[0030] A fourth aspect of the present invention provides a recombinant vector containing a nucleic acid molecule as described in the second aspect of the present invention or a nucleic acid construct as described in the third aspect of the present invention.

[0031] In some embodiments of the present invention, the recombinant vector is a recombinant cloning vector or a recombinant expression vector.

[0032] A fifth aspect of the present invention provides a transformant comprising a nucleic acid molecule as described in the second aspect of the present invention, a nucleic acid construct as described in the third aspect of the present invention, or a recombinant vector as described in the fourth aspect of the present invention, and / or expressing phenylalanine aminolysase as described in the first aspect of the present invention.

[0033] In some embodiments of the present invention, the host cells used in the construction of the transformant are selected from Escherichia coli cells, insect cells, yeast cells, and mammalian cells.

[0034] In some embodiments of the present invention, the host cell is an Escherichia coli cell, such as BL21(DE3).

[0035] A sixth aspect of the present invention provides an enzyme preparation containing phenylalanine aminolysin as described in the first aspect of the present invention.

[0036] In some embodiments of the present invention, the enzyme preparation is a liquid preparation or a lyophilized powder.

[0037] In some embodiments of the present invention, the liquid formulation comprises a buffer selected from phosphate buffer, ammonium carbonate buffer and triethanolamine-isopropylamine buffer, wherein the pH is preferably 6 to 11, more preferably 7 to 10, and more preferably 9 to 10.

[0038] In some embodiments of the present invention, the buffer solution is an ammonium carbonate buffer solution, preferably with a concentration of 1-10M, more preferably 2-6M.

[0039] The seventh aspect of the present invention provides the use of phenylalanine aminolysin as described in the first aspect of the present invention, nucleic acid molecules as described in the second aspect of the present invention, nucleic acid constructs as described in the third aspect of the present invention, recombinant vectors as described in the fourth aspect of the present invention, transformants as described in the fifth aspect of the present invention, or enzyme preparations as described in the sixth aspect of the present invention in the preparation of compounds as shown in Formula I.

[0040]

[0041] Wherein, R is hydrogen, methyl, ethyl or halogen group.

[0042] In some embodiments of the present invention, the compound represented by Formula I is (S)-β-phenylalanine or chloro-(S)-β-phenylalanine.

[0043] In some embodiments of the present invention, cinnamic acid or p-chlorocinnamic acid is used as a substrate when preparing the compound shown in Formula I.

[0044] An eighth aspect of the present invention provides a method for preparing a compound as shown in Formula I, the method comprising the following steps:

[0045] The compound shown in Formula I is prepared by using phenylalanine aminolyase as described in the first aspect of the present invention or an enzyme preparation as described in the sixth aspect of the present invention to catalyze the contact and reaction of an amino donor with a compound shown in Formula II.

[0046]

[0047] Wherein, R is hydrogen, methyl, ethyl or halogen group.

[0048] In some embodiments of the present invention, the compound represented by Formula I is (S)-β-phenylalanine, the compound represented by Formula II is cinnamic acid, or the compound represented by Formula I is chloro-(S)-β-phenylalanine, and the compound represented by Formula II is p-chlorocinnamic acid.

[0049] In this invention, an amino donor refers to a substance capable of providing amino groups to the reactants; preferably, the amino donor is a buffer solution containing ammonia, such as ammonium carbonate or ammonium phosphate, which provides a suitable pH for the reaction and allows the ammonia to serve as a source of amino groups in the reaction. In some specific embodiments of this invention, the amino donor is an ammonium carbonate buffer solution.

[0050] In some embodiments of the present invention, the mass of the phenylalanine aminolysin is 1-50% of the mass of the compound shown in Formula II, preferably 10-40%, more preferably 15-30%.

[0051] In some embodiments of the present invention, the method involves reacting the phenylalanine aminolysin with the compound shown in Formula II in the presence of a cosolvent to obtain the compound shown in Formula I.

[0052] In some embodiments of the present invention, the reaction conditions of the method are selected from one or more of the following:

[0053] (1) The concentration of the ammonium carbonate buffer solution is preferably 1-10M, more preferably 2-6M;

[0054] (2) The pH value of the ammonium carbonate buffer solution is 6 to 11, preferably 7 to 10, more preferably 9 to 10, and even more preferably 9 to 9.5;

[0055] (3) The reaction temperature is 10℃~65℃, preferably 40℃~60℃, more preferably 50℃~55℃;

[0056] (4) The reaction time is 0.1 to 120 hours, preferably 0.5 to 48 hours, and more preferably 10 to 24 hours;

[0057] (5) The co-solvent is dimethyl sulfoxide, alcohol solvent, toluene or a combination thereof, preferably dimethyl sulfoxide.

[0058] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0059] The reagents and raw materials used in this invention are all commercially available.

[0060] The positive and progressive effects of this invention are as follows:

[0061] This invention uses a mutant of phenylalanine amino acid synthase from Streptomyces maritimus as a substrate to prepare S-type β-amino acids, with significantly improved regioselectivity, enantioselectivity and conversion rate of the product. Attached Figure Description

[0062] Figure 1 This is the high-performance liquid chromatography (HPLC) chromatogram after the method of Example 1 was converted. The peak at T = 2.457 is the byproduct L-phenylalanine; t = 2.748 is the target compound (s)-β-phenylalanine; and t = 3.869 is the substrate cinnamic acid.

[0063] Figure 2 This is the high-performance liquid chromatography (HPLC) chromatogram after the method of Example 2 was converted. T = 2.743 represents the target compound (s)-β-phenylalanine; t = 3.848 represents the substrate cinnamic acid.

[0064] Figure 3 This is the high-performance liquid chromatography (HPLC) chromatogram after the method of Example 3 was converted. T = 2.741 represents the target compound (s)-β-phenylalanine; t = 3.853 represents the substrate cinnamic acid.

[0065] Figure 4 This is the high-performance liquid chromatography (HPLC) chromatogram after the method of Example 4 was converted. The peak at t = 1.61 represents the target compound, p-chloro-(s)-β-phenylalanine. Detailed Implementation

[0066] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0067] Preparation Example

[0068] Using the sequence of SEQ ID NO:1 (Gene ID: AAF81735.1, whose coding sequence is shown in SEQ ID NO:4) as the parent, directed evolution was performed using strategies such as roller PCR, iterative saturation mutagenesis, and combinatorial mutagenesis. The mutants were then transformed into *E. coli* BL21(DE3) competent cells and evenly spread on LB agar plates containing 50 μg / ml kanamycin, and incubated statically at 37°C for 18 h. The mutants on the transformed plates were then picked up with toothpicks and transferred to 96-well plates, and incubated overnight at 37°C and 220 rpm. 50 μL of bacterial culture was aspirated from the wells of the primary plate and inoculated into the corresponding wells of the secondary plate. After incubation at 37°C and 220 rpm for 2–3 h, IPTG was added to a final concentration of 0.1 mM, and the plates were incubated at 25°C for 20 h to obtain the corresponding mutants for high-throughput screening. HPLC and SFC were used for secondary screening to identify mutants with significantly improved activity and stability for gene sequencing. The sequencing results are shown in SEQ ID NO:2 and SEQ ID NO:3, and their coding sequences are shown in SEQ ID NO:5 and SEQ ID NO:6.

[0069] SEQ ID NO:1, Wild-type phenylalanine aminolysase

[0070] MTFVIELDMNVTLDQLEDAARQRTPVELSAPVRSRVRASRDVLVKFVQDERVIYGVNTSMGGFVDHLVPVSQARQLQENLINAVATNVGAYLDDTTARTIMLSRIVSLARGNSAITPANLDKLVAVLNAG IVPCIPEKGSLGTSGDLGPLAAIALVCAGQWKARYNGQIMPGRQALSEAGVEPMELSYKDGLALINGTSGMVGLGTMVLQAARRLVDRYLQVSALSVEGLAGMTKPFDPRVHGVKPHRGQRQVASRLWEGL ADSHLAVNELDTEQTLAGEMGTVAKAGSLAIEDAYSIRCTPQILGPVVDVLDRIGATLQDELNSSNDNPIVLPEEAEVFHNGHFHGQYVAMAMDHLNMALATVTNLANRRVDRFLDKSNSNGLPAFLCRED PGLRLGLMGGQFMTASITAETRTLTIPMSVQSLTSTADFQDIVSFGFVAARRAREVLTNAAYVVAFELLCACQAVDIRGADKLSSFTRPLYERTRKIVPFFDRDETITDYVEKLAADLIAGEPVDAAVAAH

[0071] SEQ ID NO:2, Phenylalanine ammonia-lyase mutant

[0072] MTFVIELDMNVTLDQLEDAARQRTPVELSAPVRSRVRASRDVLVKFVQDERVIYGVNTSMGGFVDHLVPVSQARQLQENLINAVATNVGAYLDDTTARTIMLSRIVSLARGNSAITPANLDKLVAVLNAGIVPCIPEKGSLGTSGDTGPLAAIALVCAGQWKARYNGQIMPGRQALSEAGVEPMELSYKDGLALINGTSGMVGLGTMVLQAARRLVDRYLQVSALSVEGLAGMTKPFDPRVHGLKPHRGQRQVASRLWEGLADSHLAVNELDTEQTLAGEMGTVAKAGSLAIEDAYSIRCTPQILGPVVDVLDRIGATLQDELNSSNDNPIVLPEEAEVFHNGHFHGQYVAMAMDHLNMALATVTNLANRRVDRFLDKSNSNGLPAFLCREDPGLRLGLMGGQFMTASITAETRTLTIPMSVQSLTSTADFQDIVSFGFVAARRAREVLTNAAYVVAFELLCACQAVDIRGADKLSSFTRPLYERTRKIVPFFDRDETITDYVEKLAADLIAGEPVDAAVAAH

[0073] SEQ ID NO:3, Phenylalanine ammonia-lyase mutant

[0074] MTFVIELDMNVTLDQLEDAARQRTPVELSAPVRSRVRASRDVLVKFVQDERVIYGVNTSMGGFVDHLVPVSQARQLQENLINAVATNVGAYLDDTTARTIMLSRIVSLARGNSAITPANLDKLVAVLNAGIVPCIPEKGSLGTSGDTGPLAAIALVCAGQWKARYNGQIMPGRQALSEAGVEPMELSYKDGLALINGTSGMVGLGTMVLQAARRLVDRYLQVSALSVEGLAGMTKPFDPRVHGLKPHRGQRQVASRLWEGLADSHLAVNELDTEQTLAGEMGTVAKAGSLAIEDAYSIRCTPQILGPVVDVLDIIGATLQDELNSSNDNPIVIPEEAEVFHSGHFHGQYVAMAMDHLNMALATVTNLANRRVDRFLDKSNSNGLPAFLCREDPGLRLGLMGGQFMTASITAETRTLTIPMSVQSLTSTADFQDIVSFGFVAARRAREVLTNAAYVVAFELLCACQAVDIRGADKLSSFTRPLYERTRKIVPFFDRDETITDYVEKLAADLIAGEPVDAAVAAH

[0075] SEQ ID NO:4, Coding sequence of wild-type phenylalanine ammonia-lyase

[0076]

[0077] SEQ ID NO: 5, coding sequence of a phenylalanine ammonia-lyase mutant

[0078]

[0079] SEQ ID NO:6, coding sequence of phenylalanine aminolysase mutant

[0080]

[0081] Example 1

[0082] To prepare a 4M ammonium carbonate buffer solution: Weigh 384.3g of ammonium carbonate into a 1L glass bottle. Add 900mL of purified water to the bottle and stir at room temperature until the solid dissolves. Add 35% HCl to adjust the pH to 9.5. Add purified water to bring the volume to 1L.

[0083] Weigh 112 mg of the substrate cinnamic acid and dissolve it thoroughly in 1400 μL of dimethyl sulfoxide (DMSO) until the solution is clear. Label this solution as R1. Weigh 16 mg of lyophilized phenylalanine aminolysase (amino acid sequence as shown in SEQ ID NO:1), add 3 mL of 4M ammonium carbonate buffer, and mix thoroughly until completely dissolved. Label this solution as R2. Add 2 mL of 4M ammonium carbonate buffer to an 8 mL reaction flask, then add 200 μL of R1 and 200 μL of R2. Finally, bring the volume to 4 mL with 4M ammonium carbonate buffer. Control the reaction temperature at 55℃ and shake at 1000 rpm for 18 hours to allow the reaction to proceed fully. After the reaction is complete, the conversion rate and regioselectivity are determined by HPLC. The calculation methods are as follows:

[0084]

[0085] The conversion rate was 4.65%, and the regioselectivity was 69.83%. The HPLC detection conditions were as follows: the detection column was an Agilent Poroshell 120-Chiral-T, 4.6 × 150 mm, 2.7 μm; the detection wavelength was 210 nm; the detection temperature was 35 °C; mobile phase A was 20 mM formic acid aqueous solution (pH 3.5); mobile phase B was methanol, and the ratio of mobile phase A to B was 85:15. The flow rate of the mobile phase was 1 mL / min. Figure 1 The peak at t = 2.748 indicates that the target compound is (s)-β-phenylalanine.

[0086] Example 2

[0087] To prepare a 4M ammonium carbonate buffer solution: Weigh 384.3g of ammonium carbonate into a 1L glass bottle. Add 900mL of purified water to the bottle and stir at room temperature until the solid dissolves. Add 35% HCl to adjust the pH to 9.5. Add purified water to bring the volume to 1L.

[0088] Weigh 112 mg of the substrate cinnamic acid and dissolve it thoroughly in 1400 μL of dimethyl sulfoxide (DMSO) until the solution is clear. Label this solution as R1. Weigh 16 mg of lyophilized phenylalanine aminolysase mutant 2 (amino acid sequence as shown in SEQ ID NO: 2), add 3 mL of 4M ammonium carbonate buffer, and mix thoroughly until completely dissolved. Label this solution as R2. Add 2 mL of 4M ammonium carbonate buffer to an 8 mL reaction flask, then add 200 μL of R1 and 200 μL of R2. Finally, bring the volume to 4 mL with 4M ammonium carbonate buffer. Control the reaction temperature at 55°C and shake at 1000 rpm for 18 hours to allow the reaction to proceed fully. After the reaction is complete, HPLC analysis (detection conditions as in Example 1) showed a conversion rate of 12.71% and a regioselectivity of 99.8%. Figure 2 The peak at t = 2.748 indicates that the target compound is (s)-β-phenylalanine.

[0089] Example 3

[0090] To prepare a 4M ammonium carbonate buffer solution: Weigh 384.3g of ammonium carbonate into a 1L glass bottle. Add 900mL of purified water to the bottle and stir at room temperature until the solid dissolves. Add 35% HCl to adjust the pH to 9.5. Add purified water to bring the volume to 1L.

[0091] Weigh 112 mg of the substrate cinnamic acid and dissolve it thoroughly in 1400 μL of dimethyl sulfoxide (DMSO) until the solution is clear. Label this as R1. Weigh 16 mg of lyophilized phenylalanine aminolysase mutant 3-8 and add 3 mL of 4M ammonium carbonate buffer. Mix the lyophilized powder until completely dissolved and label this as R2. Add 2 mL of 4M ammonium carbonate buffer to an 8 mL reaction flask, then add 200 μL of R1 and 200 μL of R2. Finally, bring the volume to 4 mL with 4M ammonium carbonate buffer. Control the reaction temperature at 55℃ and shake at 1000 rpm for 18 hours to allow the reaction to proceed fully. After the reaction is complete, the conversion rate and regioselectivity are detected by HPLC (detection conditions are the same as in Example 1). The results are shown in Table 1 below. The results show that mutant 8 has the best performance, and its HPLC chromatogram is shown below. Figure 3 As shown, the peak at t = 2.748 represents the target compound (s)-β-phenylalanine.

[0092] Table 1. Mutation status, transformation rate, and regioselectivity results

[0093]

[0094]

[0095] Example 4

[0096] To prepare a 4M ammonium carbonate buffer solution: Weigh 384.3g of ammonium carbonate into a 1L glass bottle. Add 900mL of purified water to the bottle and stir at room temperature until the solid dissolves. Add 35% HCl to adjust the pH to 9.5. Add purified water to bring the volume to 1L.

[0097] Weigh 64 mg of the substrate p-chlorocinnamic acid and dissolve it thoroughly in 800 μL of dimethyl sulfoxide (DMSO) until the solution is clear. Label this solution as R1. Weigh 16 mg of lyophilized powder of wild-type phenylalanine aminolysase and mutant 8 (amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:3), respectively, and add 3 mL of 4M ammonium carbonate buffer to each until completely dissolved. Label this solution as R2. Add 2 mL of 4M ammonium carbonate buffer to an 8 mL reaction flask, then add 200 μL of R1 and 200 μL of R2. Finally, bring the volume to 4 mL with 4M ammonium carbonate buffer. Control the reaction temperature at 55℃ and shake on a shaker at 1000 rpm for 18 hours to allow the reaction to proceed fully. After the reaction is complete, HPLC analysis shows that the conversion rate of wild-type is 5.5% and the regioselectivity is 77.3%. The conversion rate of mutant 8 is 20.71% and the regioselectivity is 85.4%. The HPLC detection conditions were as follows: the detection column was a Poroshell HPH C18, 4.6 × 50 mm, 2.7 μm; the detection wavelength was 220 nm; the detection temperature was 30 °C; mobile phase A was a 10 mM mixture of ammonium acetate aqueous solution and acetonitrile (water:acetonitrile = 95:5); mobile phase B was a 10 mM mixture of ammonium acetate aqueous solution and acetonitrile (water:acetonitrile = 5:95), with a ratio of 1:1 between mobile phases A and B. The flow rate of the mobile phase was 1 mL / min. Figure 4 The peak at t = 1.61 indicates that the target compound is p-chloro-(s)-β-phenylalanine.

[0098] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A phenylalanine aminolysinase, characterized in that, The amino acid sequence of the phenylalanine aminolysin differs from that shown in SEQ ID NO:1 by one or more amino acid residues at the following sites: positions 147, 244, 251, 314, 333, and 342.

2. The phenylalanine aminolysase according to claim 1, characterized in that, The phenylalanine aminotransferase, compared with the amino acid sequence shown in SEQ ID NO:1, contains one or more amino acid residues selected from the following differences: L147T, V244L, R251T, R314I / S / M, L333I / P, and N342S. Preferably, the phenylalanine amino lyase, compared with the amino acid sequence shown in SEQ ID NO:1, comprises the following amino acid residue differences: L147T and V244L, and preferably further comprises one or more of the following amino acid residue differences: R251T, R314I / S / M, L333I / P and N342S. More preferably, the phenylalanine amino lyase, compared with the amino acid sequence shown in SEQ ID NO:1, contains the following amino acid residue differences: L147T, V244L, and R314I / S / M; More preferably, in addition to including L147T, V244L, and R314I, it also includes one or more amino acid residue differences selected from the following: L333I / P and N342S; or, in addition to including L147T, V244L, and R314M, it also includes R251T. More preferably, the phenylalanine amino lyase, compared with the amino acid sequence shown in SEQ ID NO:1, includes the following amino acid residue differences: L147T, V244L, R314I and N342S, and preferably also includes the following amino acid residue differences: L333I / P.

3. The phenylalanine aminolysinase as described in claim 1, characterized in that, The phenylalanine aminotransferase, compared with the amino acid sequence shown in SEQ ID NO:1, contains one of the following combinations of amino acid residue differences: (1) L147T and V244L; (2) L147T, V244L and R314I; (3) L147T, V244L and R314S; (4) L147T, V244L, R314M and R251T; (5) L147T, V244L, R314I and N342S; (6) L147T, V244L, R314I, L333P and N342S; (7) L147T, V244L, R314I, L333I and N342S.

4. An isolated nucleic acid molecule, characterized in that, The sequence of the nucleic acid molecule is selected from: (1) A polynucleotide sequence encoding the phenylalanine aminolyase as described in any one of claims 1-3; and (2)(1) The complementary sequence of the polynucleotide sequence described in the previous two sentences; Preferably, (1) the polynucleotide sequence is as shown in SEQ ID NO:5 or 6, and more preferably as shown in SEQ ID NO.

6.

5. A nucleic acid construct, characterized in that, The nucleic acid construct contains the nucleic acid molecule as described in claim 4; the nucleic acid construct is an expression cassette.

6. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule as described in claim 4 or the nucleic acid construct as described in claim 5; Preferably, the recombinant vector is a recombinant cloning vector or a recombinant expression vector.

7. A transformant, characterized in that, The transformant contains the nucleic acid molecule as described in claim 4, the nucleic acid construct as described in claim 5, or the recombinant vector as described in claim 6, and / or expresses the phenylalanine aminolysase as described in any one of claims 1-3; Preferably, the host cells used in the construction of the transformant are selected from Escherichia coli cells, insect cells, yeast cells, and mammalian cells; More preferably, the host cell is an Escherichia coli cell, such as BL21(DE3).

8. An enzyme preparation, characterized in that, The enzyme preparation contains phenylalanine aminolysin as described in any one of claims 1-3; Preferably, the enzyme preparation is a liquid preparation or a lyophilized powder; More preferably, the liquid formulation comprises a buffer solution selected from phosphate buffer, ammonium carbonate buffer and triethanolamine-isopropylamine buffer, wherein the pH is preferably 6 to 11, more preferably 7 to 10, and even more preferably 9 to 10; More preferably, the buffer solution is an ammonium carbonate buffer solution, and its concentration is preferably 1-10M, more preferably 2-6M.

9. The use of the phenylalanine aminolysin as described in any one of claims 1-3, the nucleic acid molecule as described in claim 4, the nucleic acid construct as described in claim 5, the recombinant vector as described in claim 6, the transformant as described in claim 7, or the enzyme preparation as described in claim 8 in the preparation of compounds as shown in Formula I; in, R is hydrogen, methyl, ethyl or halogen group; Preferably, the compound represented by Formula I is (S)-β-phenylalanine or chloro-(S)-β-phenylalanine; More preferably, cinnamic acid or p-chlorocinnamic acid is used as a substrate when preparing the compound shown in Formula I.

10. A method for preparing a compound as shown in Formula I, characterized in that, The method includes the following steps: The compound of Formula I is prepared by catalyzing the amino donor with the compound of Formula II using the phenylalanine aminolyase as described in any one of claims 1-3 or the enzyme preparation as described in claim 8; Wherein, R is hydrogen, methyl, ethyl or halogen group; Preferably, the compound represented by Formula I is (S)-β-phenylalanine, the compound represented by Formula II is cinnamic acid, or the compound represented by Formula I is chloro-(S)-β-phenylalanine, the compound represented by Formula II is p-chlorocinnamic acid; and / or, the amino donor is ammonium carbonate buffer. More preferably, the mass of the phenylalanine aminolysin is 1-50% of the mass of the compound shown in Formula II, more preferably 10-40%, and even more preferably 15-30%.

11. The method as described in claim 10, characterized in that, The method involves reacting the phenylalanine aminotransferase with the compound shown in Formula II in the presence of a cosolvent to obtain the compound shown in Formula I. Preferably, the reaction conditions of the method 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 to 11, preferably 7 to 10, more preferably 9 to 10, and even more preferably 9 to 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, and more preferably 10 to 24 hours; (5) The co-solvent is dimethyl sulfoxide, alcohol solvent, toluene or a combination thereof, preferably dimethyl sulfoxide.

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