Phenylalanine ammonia mutase and application thereof in preparation of R-type beta-amino acid

Abstract

The invention discloses phenylalanine ammonia mutase and application of the phenylalanine ammonia mutase in preparation of R-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 mutase comprises the amino acid residue difference of one or more of the following sites: the 59th site, the 60th site, the 144th site, the 233th site, the 247th site, the 281th site, the 304th site, the 330th site, the 394th site, the 396th site, the 399th site, the 403th site and the 427th site. The (R)-beta-phenylalanine derivative is synthesized by adopting the enzyme, so that the regioselectivity, enantiomer selectivity and conversion rate of the product can be obviously improved.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry, and particularly relates to phenylalanine aminotransferase and its application in the preparation of R-type β-amino acids. Background Technology

[0002] Aminolysin (EC 4.3.1.X) has the function of reversibly catalyzing the cleavage of CN bonds, catalyzing the formation of ammonia and corresponding unsaturated or cyclic derivatives from α- and β-amino acid skeletons (Turner N J. Curr Opin Chem Biol. 15:234-240

[2011] ).

[0003] Phenylalanine aminotransferase (PAL), along with histidine aminotransferase (HAL) and tyrosine aminotransferase (TAL), is a member of the aromatic amino acid lyase family (EC 4.3.1.23-1.25 and 4.3.1.3). Like other aminotransferases, PAL protein structure 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. ChemCatChem. 10: 2627-2633

[2018] ).

[0004] 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] ).

[0005] However, naturally derived enzymes tend to exhibit better activity under relatively mild natural conditions, while their activity decreases under more extreme industrial reaction conditions, especially when the catalytic substrate is not a natural substrate. Engineered PAL variants have been reported to be active against cinnamic acid with small substituents on the aromatic ring (see, for example, Gloge et al. Chem. Eur. J., 6:3386-3390

[2000] ). The PAL used in this invention is derived from *Chondromyces crocatus*, and the substrate used is trans-cinnamic acid. To further realize the industrial application of this enzyme in this reaction, this invention optimizes the enzyme to provide enhanced catalytic activity. Summary of the Invention

[0006] To address the aforementioned technical problems in the prior art, this invention provides phenylalanine aminotransferase and its application in the preparation of R-type β-amino acids.

[0007] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0008] A first aspect of the present invention provides a phenylalanine ammonia mutase, wherein the amino acid sequence of the phenylalanine ammonia mutase differs from the amino acid sequence shown in SEQ ID NO:1 by one or more amino acid residues contained at the following sites: positions 59, 60, 144, 233, 247, 281, 304, 330, 394, 396, 399, 403, and 427.

[0009] In some embodiments of the present invention, the amino acid sequence of the phenylalanine aminotransferase, compared with the amino acid sequence shown in SEQ ID NO:1, includes one or more differences selected from the following amino acid residues:

[0010] (1) E59L, L144V, P233C, Q247L, L304A, L394R, C396G / Q / P / R, E399D, Y403F / L / V / T and / or N427G;

[0011] (2) L60G / S / N, G281C, L304F and / or L330M.

[0012] In “C396G / Q / P / R”, “Y403F / L / V / T”, and “L60G / S / N”, the “ / ” indicates that there is an “or” relationship between the mutable amino acid residues at the same site. The same applies to other sites below.

[0013] In some embodiments of the present invention, the amino acid sequence of the phenylalanine ammonia mutase, compared with the amino acid sequence shown in SEQ ID NO:1, includes the following amino acid residue difference: E399D, preferably further including one or more amino acid residue differences selected from the following: E59L, L60G / S / N, L144V, P233C, G281C, L304F, L330M, and C396R; preferably, the amino acid sequence of the phenylalanine ammonia mutase, compared with the amino acid sequence shown in SEQ ID NO:1, includes one of the following combinations of amino acid residue differences:

[0014] (1) E399D and L144V;

[0015] (2) E399D, L144V and C396R;

[0016] (3) E399D, L144V and E59L;

[0017] (4) E399D, G281C and E59L;

[0018] (5) E399D and G281C.

[0019] In some embodiments of the present invention, the amino acid sequence of the phenylalanine aminotransferase, compared with the amino acid sequence shown in SEQ ID NO:1, includes the following combinations of amino acid residue differences: E399D, L144V, and C396R, and also includes one or more of the following amino acid residue differences: L60G / S / N and L330M.

[0020] In some embodiments of the present invention, the amino acid sequence of the phenylalanine aminotransferase, compared with the amino acid sequence shown in SEQ ID NO:1, includes the following combinations of amino acid residues that differ: E399D, L144V, C396R, and L60N; or, E399D, L144V, C396R, and L60S.

[0021] A second aspect of the present invention provides an isolated nucleic acid molecule, the sequence of which is selected from:

[0022] (1) A polynucleotide sequence encoding the phenylalanine aminotransferase as described in the first aspect of the present invention; and

[0023] (2)(1) The complementary sequence of the polynucleotide sequence.

[0024] In some embodiments of the present invention, (1) the polynucleotide sequence is shown as SEQ ID NO:6, 7 or 8, preferably as shown as SEQ ID NO:8.

[0025] 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.

[0026] 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.

[0027] In some embodiments of the present invention, the recombinant vector is a recombinant cloning vector or a recombinant expression vector.

[0028] In some embodiments of the present invention, the skeleton of the recombinant vector is pET-30a.

[0029] 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 a phenylalanine aminotransferase as described in the first aspect of the present invention.

[0030] 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.

[0031] In some embodiments of the present invention, the host cell is Escherichia coli, such as BL21(DE3).

[0032] A sixth aspect of the present invention provides an enzyme preparation containing phenylalanine aminotransferase as described in the first aspect of the present invention.

[0033] The seventh aspect of the present invention provides the use of the phenylalanine aminotransferase as described in the first aspect of the present invention, the nucleic acid molecule as described in the second aspect of the present invention, the nucleic acid construct as described in the third aspect of the present invention, the recombinant vector as described in the fourth aspect of the present invention, the transformant as described in the fifth aspect of the present invention, or the enzyme preparation as described in the sixth aspect of the present invention in the preparation of compounds as shown in Formula I.

[0034]

[0035] Where R is hydrogen, halogen, or hydroxyl.

[0036] In some embodiments of the present invention, the compound represented by Formula I is (R)-3-amino-3-phenylpropionic acid or (R)-3-amino-3-p-chlorophenylpropionic acid.

[0037] In some embodiments of the present invention, when preparing the compound shown in Formula I, trans-cinnamic acid or p-chlorocinnamic acid is used as a substrate.

[0038] 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:

[0039] The compound shown in Formula I is prepared by using the phenylalanine aminotransferase as described in the first aspect of the present invention or the enzyme preparation as described in the sixth aspect of the present invention to catalyze the contact and reaction of the amino donor with the compound shown in Formula II.

[0040]

[0041]

[0042] Where R is hydrogen, halogen, or hydroxyl.

[0043] In some embodiments of the present invention, the compound represented by Formula I is (R)-3-amino-3-phenylpropionic acid or (R)-3-amino-3-p-chlorophenylpropionic acid; and the compound represented by Formula II is trans-cinnamic acid or p-chlorocinnamic acid.

[0044] In some embodiments of the present invention, the amino donor is an ammonium carbonate buffer solution.

[0045] In some embodiments of the present invention, the mass of the phenylalanine aminotransferase is 3.7-11.1 times the weight of the compound shown in Formula I.

[0046] In some embodiments of the present invention, the reaction conditions of the method are selected from one or more of the following:

[0047] (1) The pH of the reaction is 6.0 to 10.0, preferably 8.0 to 10.0, and more preferably 9.5;

[0048] (2) The reaction temperature is 10℃~50℃, preferably 20℃~45℃, and more preferably 30℃;

[0049] (3) The reaction time is 0.1 to 48 hours, preferably 0.5 to 24 hours, and more preferably 10 to 24 hours;

[0050] (4) The concentration of the ammonium carbonate buffer solution is 2-6 mol / L, for example 4 mol / L.

[0051] 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.

[0052] The reagents and raw materials used in this invention are all commercially available.

[0053] The positive and progressive effects of this invention are as follows:

[0054] The enzyme obtained by the mutation optimization of phenylalanine aminotransferase in this invention can be used to synthesize (R)-β-phenylalanine derivatives, which can significantly improve the regioselectivity, enantioselectivity and conversion rate of the product. Attached Figure Description

[0055] Figure 1 This is the high-performance liquid chromatography (HPLC) chromatogram of (R)-3-amino-3-phenylpropionic acid after conversion by the method in Example 4. The peak at t = 2.674 represents the target compound (R)-3-amino-3-phenylpropionic acid.

[0056] Figure 2 This is the high-performance gas chromatography (HPLC) chromatogram of (R)-3-amino-3-phenylpropionic acid after conversion by the method in Example 5. The peak at t = 2.674 represents the target compound (R)-3-amino-3-phenylpropionic acid.

[0057] Figure 3 This is the high-performance liquid chromatography (HPLC) chromatogram of (R)-3-amino-3-phenylpropionic acid after conversion by the method in Example 6. The peak at t = 2.674 represents the target compound (R)-3-amino-3-phenylpropionic acid.

[0058] Figure 4 This is the high-performance liquid chromatography (HPLC) chromatogram of (R)-3-amino-3-p-chlorophenylpropionic acid after conversion by the method in Example 7. The peak at t = 1.370 represents the target compound (R)-3-amino-3-p-chlorophenylpropionic acid. Detailed Implementation

[0059] 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.

[0060] Preparation Example

[0061] Using the sequence of SEQ ID NO:1 (coding sequence as shown in SEQ ID NO:5) as the parent culture, directed evolution was performed using strategies such as roller PCR, iterative saturation mutagenesis, and combinatorial mutagenesis. The recombinant expression vector containing the mutant coding sequence was 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. Mutants from the transformed plates were selected using toothpicks and transferred to 96-well plates, and incubated overnight at 37°C and 220 rpm. 50 μL of bacterial culture was transferred from the wells of the primary plate to the corresponding wells of the secondary plate, and incubated at 37°C and 220 rpm for 2–3 h. IPTG was then added to a final concentration of 0.2 mM, and the plates were incubated at 30°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:6 and SEQ ID NO:7, respectively.

[0062] SEQ ID NO:1, coding sequence of wild-type phenylalanine aminotransferase

[0063] MKITGSNLSIYDVADVCMKRATVELDPSQLERVAVAHERTQAWGEAQHPIYGVNTGFGELVPVMIPRQHKRELQENLIRSHAAGGGEPFADDVVRAIMLARLNCLMKGYSGASVETVKLLAEFINRGIHPVIPQQGSLGASGDLSPLSHIALALIGEGTVSFKGQVRKTGDVLREEGLKPLELGFKGGLTLINGTSAMTGAACVALGRAYHLFRLALLATADFVQCLGGSTGPFEERGHLPKNHSGQVIVAREIRKLLAGSQLTSDHQDLMKEMVARSGVGNDVVDTGVYLQDAYTLRAVPQILGPVLDTLDFARKLIEEELNSTNDNPLIFDVPEQTFHGANFHGQYVAMACDYLNIAVTEIGVLAERQLNRLVDPNINGKLPPFLASAHSGLLCGFEGGQYLATSIASENLDLAAPSSIKSLPSNGSNQDVVSMGTTSARKSLRLCENVGTIVSTLIAACNQAGHILGNERFSPPIRELHGELSRSVPLYQDDSPIFELFQTVRAFVGGDGFRAHLVTHLDLAATTASS

[0064] SEQ ID NO:2, Coding sequence of phenylalanine ammonia-lyase mutant

[0065] MKITGSNLSIYDVADVCMKRATVELDPSQLERVAVAHERTQAWGEAQHPIYGVNTGFGELVPVMIPRQHKRELQENLIRSHAAGGGEPFADDVVRAIMLARLNCLMKGYSGASVETVKLLAEFINRGIHPVIPQQGSLGASGDLSPLSHIALALIGEGTVSFKGQVRKTGDVLREEGLKPLELGFKGGLTLINGTSAMTGAACVALGRAYHLFRLALLATADFVQCLGGSTGPFEERGHLPKNHSGQVIVAREIRKLLAGSQLTSDHQDLMKEMVARSGVGNDVVDTGVYLQDAYTLRAVPQILGPVLDTLDFARKLIEEELNSTNDNPLIFDVPEQTFHGANFHGQYVAMACDYLNIAVTEIGVLAERQLNRLVDPNINGKLPPFLASAHSGLLCGFDGGQYLATSIASENLDLAAPSSIKSLPSNGSNQDVVSMGTTSARKSLRLCENVGTIVSTLIAACNQAGHILGNERFSPPIRELHGELSRSVPLYQDDSPIFELFQTVRAFVGGDGFRAHLVTHLDLAATTASS

[0066] SEQ ID NO:3, Coding sequence of phenylalanine ammonia-lyase mutant

[0067] MKITGSNLSIYDVADVCMKRATVELDPSQLERVAVAHERTQAWGEAQHPIYGVNTGFGELVPVMIPRQHKRELQENLIRSHAAGGGEPFADDVVRAIMLARLNCLMKGYSGASVETVKLLAEFINRGIHPVIPQQGSLGASGDVSPLSHIALALIGEGTVSFKGQVRKTGDVLREEGLKPLELGFKGGLTLINGTSAMTGAACVALGRAYHLFRLALLATADFVQCLGGSTGPFEERGHLPKNHSGQVIVAREIRKLLAGSQLTSDHQDLMKEMVARSGVGNDVVDTGVYLQDAYTLRAVPQILGPVLDTLDFARKLIEEELNSTNDNPLIFDVPEQTFHGANFHGQYVAMACDYLNIAVTEIGVLAERQLNRLVDPNINGKLPPFLASAHSGLLRGFDGGQYLATSIASENLDLAAPSSIKSLPSNGSNQDVVSMGTTSARKSLRLCENVGTIVSTLIAACNQAGHILGNERFSPPIRELHGELSRSVPLYQDDSPIFELFQTVRAFVGGDGFRAHLVTHLDLAATTASS

[0068] SEQ ID NO:4, Coding sequence of phenylalanine ammonia-lyase mutant

[0069] MKITGSNLSIYDVADVCMKRATVELDPSQLERVAVAHERTQAWGEAQHPIYGVNTGFGENVPVMIPRQHKRELQENLIRSHAAGGGEPFADDVVRAIMLARLNCLMKGYSGASVETVKLLAEFINRGIHPVIPQQGSLGASGDVSPLSHIALALIGEGTVSFKGQVRKTGDVLREEGLKPLELGFKGGLTLINGTSAMTGAACVALGRAYHLFRLALLATADFVQCLGGSTGPFEERGHLPKNHSGQVIVAREIRKLLAGSQLTSDHQDLMKEMVARSGVGNDVVDTGVYLQDAYTLRAVPQILGPVLDTLDFARKLIEEELNSTNDNPLIFDVPEQTFHGANFHGQYVAMACDYLNIAVTEIGVLAERQLNRLVDPNINGKLPPFLASAHSGLLRGFDGGQYLATSIASENLDLAAPSSIKSLPSNGSNQDVVSMGTTSARKSLRLCENVGTIVSTLIAACNQAGHILGNERFSPPIRELHGELSRSVPLYQDDSPIFELFQTVRAFVGGDGFRAHLVTHLDLAATTASS

[0070] SEQ ID NO:5, wild-type phenylalanine ammonia-lyase

[0071]

[0072] SEQ ID NO:6, phenylalanine aminotransferase mutant

[0073]

[0074] SEQ ID NO:7, phenylalanine aminotransferase mutant

[0075]

[0076] SEQ ID NO:8, phenylalanine aminotransferase mutant

[0077]

[0078] Example 1: Expression and Screening of Engineered Phenylalanine Aminomutase

[0079] The polynucleotide sequence SEQ ID NO:5 was cloned into the pET-30a vector system and subsequently expressed in *E. coli* strain BL21(DE3). *E. coli* strain BL21(DE3) expressed phenylalanine aminotransferase polypeptides under the control of the T7 promoter. Based on sequence comparison with other phenylalanine aminotransferases and computer simulation and active site analysis of the enzyme structure of the docked substrate trans-cinnamic acid, potential activity or selectivity-related amino acid residue positions were identified, and mutagenic sites were rationally designed. First-round beneficial mutants of these enzymes were screened using trans-cinnamic acid as a substrate under HPLC conditions. Beneficial mutants with increased activity or selectivity were identified. Combinatorial mutants were constructed from beneficial mutant residues from the first round of screening in various arrangements, and improved catalytic properties were screened under HPLC conditions. The engineered phenylalanine aminotransferase polypeptide sequences and specific mutations obtained from the screening were analyzed, and their catalytic efficiencies were determined.

[0080] Example 2

[0081] As mentioned above, the engineered phenylalanine aminotransferase polypeptide of Example 1 was produced in Escherichia coli BL21(DE3) under the control of the T7 promoter. It was used for the preparation of crude enzyme solution as follows.

[0082] 1. Preparation of buffer solution

[0083] Measure 1000 mL of purified water, add 484.36 g of ammonium carbonate, and stir until completely dissolved. Maintain the pH at 9.5 and stir until the solid is completely dissolved.

[0084] 2. Cultivation, induction of expression, and preparation of crude enzyme solution product

[0085] Cells were picked and cultured overnight in LB liquid medium containing 10 mg / L glucose and 50 mg / L kanamycin at 37°C, 250 rpm, and 85% humidity. Then, 10 μL aliquots of the overnight grown cells were transferred to 390 μL of 3×TB growth medium containing 50 mg / L kanamycin and cultured at 37°C and 450 rpm for 2–3 hours (OD). 600=0.6~1.0), add IPTG to a final concentration of 0.1mM, and incubate at 25℃, 450rpm for 20h. Centrifuge the cell culture at 4000rpm, 4℃ for 12min and discard the culture medium. Place the cell clumps (i.e., wet cells) at -80℃ for 2h, then resuspend them in 270μL ammonium carbonate buffer (2mg / mL lysozyme and 1mg / mL nuclease). Lyse at 37℃ for 2h. Centrifuge at 4000rpm for 15min and collect the supernatant to prepare crude enzyme solution, in which 20mg of wet cells corresponds to 2mg of phenylalanine aminotransferase.

[0086] Example 3

[0087] In this embodiment, the analytical procedure describes the analysis of the catalytic results of the enzyme mutant provided by the present invention on trans-cinnamic acid. The method employed is HPLC analysis. In a 96-well deep plate, 1 mL of methanol was added to the reaction solution. The plate was centrifuged at 4000 rpm for 20 min. 250 μL of the supernatant was transferred into a 96-well shallow plate for HPLC analysis. The quenched reaction was analyzed by HPLC under the following conditions. The conversion of compound II to compound I was determined from the resulting spectra as follows:

[0088]

[0089] The HPLC analysis conditions for implementation cases 4, 5, and 6 are as follows: column: Agilent Poroshell 120-Chiral-T, 4.6 × 150 mm, 2.7 μm; mobile phase A: 20 mm NH4FA aqueous solution (pH = 3.5); mobile phase B: MeOH; A:B ratio: 85:15; isocratic elution; detection wavelength: 210 nm; column temperature: 35 °C; flow rate: 1 mL / min; injection volume: 1.0 μL; run time: 3.30 min.

[0090] This method can be used to rapidly identify the conversion of trans-cinnamic acid to (R)-3-amino-3-phenylpropionic acid.

[0091] The HPLC analysis conditions for Implementation Case 7 were as follows: column: Poroshell HPH C18, 4.6×50mm, 2.7μm; mobile phase A: a mixed solution of 10mM ammonium acetate in water and acetonitrile (water:acetonitrile = 95:5); mobile phase B: a mixed solution of 10mM ammonium acetate in water and acetonitrile (water:acetonitrile = 5:95), with a ratio of 1:1 between mobile phases A and B; isocratic elution; detection wavelength: 220nm; column temperature: 30℃; flow rate: 1.0ml / min; injection volume: 1μL; run time: 2.800min.

[0092] Example 4

[0093] A method for converting trans-cinnamic acid to (R)-3-amino-3-phenylpropionic acid using a crude enzyme solution of phenylalanine aminomutase.

[0094] In this embodiment, different combined mutants were constructed using WT as a template, and their catalytic activity was tested. A method for converting compound II to compound I using the crude enzyme solution prepared in Example 2 is described. The prepared crude enzyme solution was added to a 250 μL scale reaction. The reaction system consisted of: 384.36 g / L ammonium carbonate buffer, 1 g / L substrate trans-cinnamic acid, and 50 μL crude enzyme solution (wet bacterial count 3.7 mg, enzyme count 0.37 mg).

[0095] The specific steps are as follows: Add 25 mg of trans-cinnamic acid to 10 mL of ammonium carbonate buffer. Add 100 μL of the mixed solution and 50 μL of crude enzyme solution to an equal portion of a 96-well deep plate, and incubate the reaction at 30°C for 18 h. After the reaction is complete, add 1 mL of methanol. Centrifuge the plate at 4000 rpm for 20 min. Transfer 250 μL of the supernatant to a 96-well shallow plate for HPLC analysis. Determine the conversion rate using HPLC analysis; the calculation method is as follows:

[0096]

[0097] After reacting for 18 hours, HPLC analysis revealed that the optimal mutant (E399D mutant, whose amino acid sequence is shown in SEQ ID NO:2 and coding sequence is shown in SEQ ID NO:6) had a conversion rate of 2.22%. Figure 1 The peak at t = 2.674 indicates that the target compound (R)-3-amino-3-phenylpropionic acid is present.

[0098] Table 1: Mutant sequence information and catalytic results used in implementing Case 4

[0099] Variant number Variations Conversion rate (%) 1 WT 0.32 2 E399D 2.22 3 Y403L 2.00 4 Y403T 1.47 5 L304A 1.43 6 C396Q 1.34 7 C396P 1.31 8 C396R 1.20 9 Y403F 1.19 10 Y403L 1.01 11 C396G 0.91 12 Y403V 0.86 13 N427G 0.85 14 Q247L 0.72 15 L144V 0.61 16 L394R 0.59 17 E59L 0.53

[0100] Example 5

[0101] A method for converting trans-cinnamic acid to (R)-3-amino-3-phenylpropionic acid using a crude enzyme solution of phenylalanine aminomutase.

[0102] In this embodiment, different combined mutants were constructed using the E399D mutant as a template, and their catalytic activity was tested. A method for converting compound II to compound I using the crude enzyme solution prepared in Example 2 is described. The prepared crude enzyme solution was added to a 250 μL scale reaction. The reaction system consisted of: 384.36 g / L ammonium carbonate buffer, 1 g / L substrate trans-cinnamic acid, and 50 μL crude enzyme solution (wet bacterial count 3.7 mg, enzyme count 0.37 mg).

[0103] The specific steps are as follows: Add 25 mg of trans-cinnamic acid to 10 mL of ammonium carbonate buffer. Add 100 μL of the mixed solution and 50 μL of crude enzyme solution equally to a 96-well deep plate, and incubate the reaction at 30°C for 18 h. After the reaction is complete, add 1 mL of methanol. Centrifuge the plate at 4000 rpm for 20 min. Transfer 250 μL of the supernatant to a 96-well shallow plate for HPLC analysis. The conversion rate is determined by HPLC analysis, and the calculation method is as follows:

[0104]

[0105] After 18 hours of reaction, HPLC analysis revealed that the optimal mutant (E399D / L144V / C396R mutant, whose amino acid sequence is shown in SEQ ID NO:3 and coding sequence in SEQ ID NO:7) had a conversion rate of 7.67%. Figure 2 The peak at t = 2.674 indicates that the target compound (R)-3-amino-3-phenylpropionic acid is present.

[0106] Table 2: Mutant sequence information and catalytic results used in implementing Case 5

[0107] Variant number Variations Conversion rate (%) 1 E399D 2.22 2 E399D / L144V / C396R 7.67 3 E399D / L144V 4.77 4 E399D / L144V / E59L 4.36 5 E399D / G281C / E59L 2.82 6 E399D / G281C 3.32

[0108] Example 6

[0109] A method for converting trans-cinnamic acid to (R)-3-amino-3-phenylpropionic acid using a crude enzyme solution of phenylalanine aminomutase.

[0110] In this embodiment, different combined mutants were constructed using the E399D / L144V / C396R mutant as a template, and their catalytic activity was tested. A method for converting compound II to compound I using the crude enzyme solution prepared in Example 2 is described. The prepared crude enzyme solution was added to a 250 μL scale reaction. The reaction system consisted of: 384.36 g / L ammonium carbonate buffer, 1 g / L substrate compound II, and 50 μL crude enzyme solution (wet bacterial count 3.7 mg, enzyme count 0.37 mg).

[0111] The specific steps are as follows: Add 25 mg of trans-cinnamic acid to 20 mL of ammonium carbonate buffer. Add 200 μL of the mixed solution and 50 μL of crude enzyme solution equally to a 96-well deep plate, and incubate the reaction at 30°C for 18 h. After the reaction is complete, add 1 mL of methanol. Centrifuge the plate at 4000 rpm for 20 min. Transfer 250 μL of the supernatant to a 96-well shallow plate for HPLC analysis. Determine the conversion rate using HPLC analysis; the calculation method is as follows:

[0112]

[0113] After reacting for 18 hours, HPLC analysis revealed that the optimal mutant (its amino acid sequence is shown in SEQ ID NO:4, and its coding sequence is shown in SEQ ID NO:8) had a conversion rate of 41.33%. Figure 3 The peak at t = 2.674 indicates that the target compound (R)-3-amino-3-phenylpropionic acid is present.

[0114] Table 3: Mutant sequence information and catalytic results used in implementing Case 6

[0115]

[0116]

[0117] Example 7

[0118] A method for converting p-chlorocinnamic acid to (R)-3-amino-3-p-chlorophenylpropionic acid using crude enzyme solution of phenylalanine aminotransferase.

[0119] In this example, the optimal mutant E399D / L144V / C396R / L60N was used in conjunction with the wild-type catalyst for p-chlorocinnamic acid, and its catalytic activity was tested. The crude enzyme solution prepared in Example 2 was added to a 250 μL scale reaction. The reaction system consisted of: 384.36 g / L ammonium carbonate buffer, 1 g / L p-chlorocinnamic acid substrate, and 50 μL of crude enzyme solution (wet cell count 3.7 mg, enzyme count 0.37 mg).

[0120] The specific steps are as follows: Add 25 mg of p-chlorocinnamic acid to 20 mL of ammonium carbonate buffer. Add 200 μL of the mixed solution and 50 μL of crude enzyme solution to an equal portion of the reaction mixture. Incubate the reaction at 30 °C for 18 h. After the reaction is complete, add 1 mL of methanol. Centrifuge the plate at 4000 rpm for 20 min. Take 250 μL of the supernatant for HPLC analysis. Determine the catalytic efficiency through HPLC analysis.

[0121] After 18 hours of reaction, HPLC analysis showed that the mutant E399D / L144V / C396R / L60N exhibited catalytic activity compared to the wild type. Figure 4 The peak at t = 1.370 indicates that the target compound (R)-3-amino-3-p-chlorophenylpropionic acid is present.

[0122] In summary, the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A phenylalanine aminotransferase, characterized in that, The amino acid sequence of the phenylalanine ammonia mutase differs from that shown in SEQ ID NO:1 in one or more of the following amino acid residues: at positions 59, 60, 144, 233, 247, 281, 304, 330, 394, 396, 399, 403, and 427.

2. The phenylalanine aminotransferase as described in claim 1, characterized in that, The amino acid sequence of the phenylalanine aminotransferase differs from the amino acid sequence shown in SEQ ID NO:1 by one or more of the following amino acid residue differences: (1) E59L, L144V, P233C, Q247L, L304A, L394R, C396G / Q / P / R, E399D, Y403F / L / V / T and / or N427G; (2) L60G / S / N, G281C, L304F and / or L330M; Preferably, the amino acid sequence of the phenylalanine ammonia mutase, compared with the amino acid sequence shown in SEQ ID NO:1, includes the following amino acid residue difference: E399D, and preferably further includes one or more amino acid residue differences selected from the following: E59L, L60G / S / N, L144V, P233C, G281C, L304F, L330M, and C396R; preferably, the amino acid sequence of the phenylalanine ammonia mutase, compared with the amino acid sequence shown in SEQ ID NO:1, includes one of the following combinations of amino acid residue differences: (1) E399D and L144V; (2) E399D, L144V and C396R; (3) E399D, L144V and E59L; (4) E399D, G281C and E59L; (5) E399D and G281C; More preferably, the amino acid sequence of the phenylalanine aminotransferase, compared with the amino acid sequence shown in SEQ ID NO:1, includes the following amino acid residue differences: E399D, L144V and C396R, and also includes one or more amino acid residue differences selected from the following: L60G / S / N and L330M. More preferably, the amino acid sequence of the phenylalanine aminotransferase, compared with the amino acid sequence shown in SEQ ID NO:1, includes the following combinations of amino acid residues that are different: E399D, L144V, C396R, and L60N; or, E399D, L144V, C396R, and L60S.

3. 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 aminotransferase as described in claim 1 or 2; and (2)(1) The complementary sequence of the polynucleotide sequence; Preferably, (1) the polynucleotide sequence is as shown in SEQ ID NO:6, 7 or 8, and more preferably as shown in SEQ ID NO.

8.

4. A nucleic acid construct, characterized in that, The nucleic acid construct contains the nucleic acid molecule as described in claim 3; the nucleic acid construct is an expression cassette.

5. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule as described in claim 3 or the nucleic acid construct as described in claim 4; Preferably, the recombinant vector is a recombinant cloning vector or a recombinant expression vector; More preferably, the skeleton of the recombinant vector is pET-30a.

6. A transformant, characterized in that, The transformant contains the nucleic acid molecule as described in claim 3, the nucleic acid construct as described in claim 4, or the recombinant vector as described in claim 5, and / or expresses the phenylalanine aminotransferase as described in claim 1 or 2; 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 Escherichia coli, such as BL21(DE3).

7. An enzyme preparation, characterized in that, The enzyme preparation contains the phenylalanine aminotransferase as described in claim 1 or 2.

8. The use of the phenylalanine aminotransferase as described in claim 1 or 2, the nucleic acid molecule as described in claim 3, the nucleic acid construct as described in claim 4, the recombinant vector as described in claim 5, the transformant as described in claim 6, or the enzyme preparation as described in claim 7 in the preparation of compounds as shown in Formula I; in, R is hydrogen, halogen, or hydroxyl; Preferably, the compound represented by Formula I is (R)-3-amino-3-phenylpropionic acid or (R)-3-amino-3-p-chlorophenylpropionic acid; More preferably, when preparing the compound shown in Formula I, trans-cinnamic acid or p-chlorocinnamic acid is used as the substrate.

9. 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 aminotransferase as described in claim 1 or 2 or the enzyme preparation as described in claim 7. Wherein, R is hydrogen, halogen, or hydroxyl; Preferably, the compound of Formula I is (R)-3-amino-3-phenylpropionic acid or (R)-3-amino-3-p-chlorophenylpropionic acid; the compound of Formula II is trans-cinnamic acid or p-chlorocinnamic acid; More preferably, the amino donor is an ammonium carbonate buffer solution; More preferably, the mass of the phenylalanine aminotransferase is 3.7-11.1 times the weight of the compound shown in Formula I.

10. The method as described in claim 9, characterized in that, The reaction conditions for the method are selected from one or more of the following: (1) The pH of the reaction is 6.0 to 10.0, preferably 8.0 to 10.0, and more preferably 9.5; (2) The reaction temperature is 10℃~50℃, preferably 20℃~45℃, and more preferably 30℃; (3) The reaction time is 0.1 to 48 hours, preferably 0.5 to 24 hours, and more preferably 10 to 24 hours; (4) The concentration of the ammonium carbonate buffer solution is 2-6 mol / L, for example 4 mol / L.

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