Phenylalanine ammonia-lyase mutant and application thereof

Abstract

The phenylalanine ammonia-lyase mutant is a mutant in which asparagine (N) at position 272 and / or tyrosine (Y) at position 365 in the sequence shown in SEQ ID NO: 2 is mutated into lysine (K) and phenylalanine (F) respectively. The phenylalanine ammonia-lyase mutant can catalyze the preparation of L-phenylalanine compounds from high-concentration cinnamic acid compounds, solve the problem of substrate inhibition in catalytic reaction, thereby reduce the production cost and meet the industrialization demand.

Description

A phenylalanine ammonia-lyase mutant and its application Technical Field

[0001] The invention belongs to the field of biological enzyme catalysis and relates to a phenylalanine ammonia lyase mutant. Background Art

[0002] L-phenylalanine compounds, including L-phenylalanine and its derivatives, are widely used in the pharmaceutical field. For example, L-phenylalanine derivatives such as 3-bromophenyl-L-alanine and (S)-2-chlorophenylalanine are important pharmaceutical intermediates. (S)-2-chlorophenylalanine is the primary raw material for the production of perindopril, an angiotensin I-converting enzyme (ACE) inhibitor, a drug for the treatment of hypertension.

[0003] Existing technologies for producing L-phenylalanine compounds mainly include immobilized cell biotransformation, microbial fermentation, chemical synthesis, and bioenzymatic methods, among which the bioenzymatic method has the greatest application prospects. Patent document WO2006 / 069799A1 discloses a process for preparing (S)-2-amino-3-(2-chloro-phenyl)propionic acid using phenylalanine ammonia lyase (PAL) from the red yeast Rhodotorula glutinis to catalyze the reaction of 3-(2-chloro-phenyl)acrylic acid with aqueous ammonia. However, this process suffers from substrate inhibition (i.e., the initial enzyme activity decreases with increasing substrate concentration), requiring operation at low concentrations. Consequently, large volumes or complex feeding schemes are required to produce acceptable amounts of product. Patent document CN101517089A provides a method for catalyzing the reaction of cinnamic acid compounds with amino donors using phenylalanine ammonia lyase derived from the hydrothermal marine bacterium Idiomarina loihiensis. Compared with yeast phenylalanine ammonia lyase, substrate inhibition is significantly improved, but as the substrate concentration increases to 100mM, the increase in the initial enzyme activity decreases.

[0004] Therefore, it is necessary to develop an enzyme that can catalyze higher concentrations of cinnamic acid compound substrates to prepare L-phenylalanine compounds to meet industrial needs. Summary of the Invention

[0005] The purpose of the present invention is to provide a mutant phenylalanine ammonia lyase with higher activity to solve the problems existing in the prior art.

[0006] In a first aspect, the present invention provides a phenylalanine ammonia lyase mutant, wherein the asparagine (N) at position 272 of the sequence shown in SEQ ID NO: 2 is mutated to lysine (K) and / or the tyrosine (Y) at position 365 is mutated to phenylalanine (F).

[0007] In a preferred embodiment, the phenylalanine ammonia lyase mutant is PAL-K (amino acid sequence as shown in SEQ ID NO: 7), PAL-F (amino acid sequence as shown in SEQ ID NO: 8) or PAL-KF (amino acid sequence as shown in SEQ ID NO: 9).

[0008] In a second aspect, the present invention provides a nucleic acid molecule encoding the phenylalanine ammonia lyase mutant.

[0009] Those skilled in the art can design a suitable nucleic acid molecule based on the amino acid sequence of the phenylalanine ammonia lyase mutant disclosed in the present invention and the vector used.

[0010] In a preferred embodiment, the sequence of the nucleic acid molecule is compared with SEQ ID NO: 1, and bases 814 to 816 are AAG and / or bases 1093 to 1095 are TTC.

[0011] In a third aspect, the present invention provides a recombinant vector comprising the above-mentioned nucleic acid molecule.

[0012] In some embodiments, the starting vector of the recombinant vector may be a replication vector or an expression vector, and the expression vector may be a pET series vector, a shuttle vector, a phage or a viral vector.

[0013] In a preferred embodiment, the starting vector of the recombinant vector is pET28a(+).

[0014] In a fourth aspect, the present invention provides a recombinant cell comprising the aforementioned nucleic acid molecule or recombinant vector.

[0015] In some embodiments, the starting cell of the recombinant cell can be any cell capable of expressing a protein.

[0016] In a preferred embodiment, the recombinant cell is a genetically engineered bacterium, and the starting bacterium is E. coli BL21 (DE3).

[0017] In a fifth aspect, the present invention provides a composition comprising the phenylalanine ammonia lyase mutant of the present invention.

[0018] In a sixth aspect, the present invention provides the use of the phenylalanine ammonia lyase mutant, nucleic acid molecule, recombinant vector, recombinant cell, and composition in the synthesis of L-phenylalanine compounds.

[0019] In a preferred embodiment, the L-phenylalanine compound is (S)-2-chlorophenylalanine.

[0020] In a seventh aspect, the present invention provides a method for synthesizing an L-phenylalanine-like compound, comprising the steps of adding a fermentation culture of a phenylalanine ammonia-lyase mutant or recombinant cell of the present invention, or a composition of the present invention, to a reaction system containing a substrate and an amino donor solution, wherein the substrate is trans-2-chloro-cinnamic acid and the amino donor solution is ammonia (NH3) or an aqueous ammonia solution. The L-phenylalanine-like compound is (S)-2-chlorophenylalanine.

[0021] In a preferred embodiment, the fermentation culture of the recombinant cells can be wet cells obtained through fermentation culture, or a crude enzyme solution extracted after ultrasonic disruption of the wet cells, or a purified pure enzyme solution.

[0022] In a preferred embodiment, the amino donor solution is aqueous ammonia, and the concentration of the aqueous ammonia is in the range of 3 to 6M, preferably 3 to 4M.

[0023] In a preferred embodiment, in the method, the weight-to-volume ratio of the trans-2-chloro-cinnamic acid to the amino donor solution is 20-50 g / L, preferably 40-50 g / L.

[0024] In a preferred embodiment, the weight ratio of trans-2-chloro-cinnamic acid to wet bacteria is 0.5-4:1, preferably 1-3:1, and most preferably 2:1.

[0025] The wet bacterial cells of the present invention are obtained by inducing and culturing recombinant cells, separating the culture fluid to obtain bacterial cells containing the recombinant phenylalanine ammonia lyase mutant, and separating the culture fluid and the bacterial cells to obtain the wet bacterial cells.

[0026] In a preferred embodiment, the method comprises: mixing wet cells obtained by fermentation culture of the recombinant cells of the present invention, or crude enzyme solution extracted after ultrasonic disruption of wet cells, or purified pure enzyme solution with half the amount of aqueous ammonia, placing the mixture in a reactor and reacting at 30°C, mixing the other half of aqueous ammonia and trans-2-chloro-cinnamic acid to prepare a feed, feeding the feed at a rate of 125ml to 200mL / h, and continuing the conversion for 5h after the feeding is completed, to obtain the product. In the entire method, the weight-to-volume ratio of the trans-2-chloro-cinnamic acid to the amino donor solution is 20 to 50g / L, preferably 40 to 50g / L; the weight ratio of trans-2-chloro-cinnamic acid to the wet cells is 0.5 to 4:1, preferably 1 to 3:1, and most preferably 2:1; and the concentration of the aqueous ammonia is 3 to 6M, preferably 3 to 4M.

[0027] The dosage of crude enzyme solution and pure enzyme solution can be calculated according to the dosage of wet bacteria.

[0028] In a preferred embodiment, the method for preparing wet cells obtained by fermentation culture of the recombinant cells of the present invention is as follows: the recombinant cells are inoculated into LB liquid culture medium containing a final concentration of 50 μg / mL kanamycin resistance, cultured at 37°C, 180 rpm for 10 hours, and then inoculated into fresh LB liquid culture medium containing a final concentration of 50 μg / mL kanamycin resistance at a volume concentration of 2%. The cells are cultured at 37°C, 180 rpm until the cell OD600 reaches 0.8-1.0, IPTG is added at a final concentration of 0.1 mM, and after induction culture at 28°C for 12 hours, the cells are centrifuged at 4°C, 6000 rpm for 10 minutes, and the supernatant is removed to obtain wet cells.

[0029] In a preferred embodiment, the crude enzyme solution of the present invention is prepared by adding 9.8 mL of sodium phosphate buffer solution to 0.2 g of wet bacterial cells, resuspending the wet cells in 100 mM sodium phosphate buffer, pH 8.0, and ultrasonically disrupting the cells in an ice bath to obtain a cell lysate. The cell lysate is centrifuged at 6000 rpm and 4°C for 10 minutes to obtain a supernatant, which is the crude enzyme solution. The crude enzyme solution is then purified to obtain a pure enzyme solution.

[0030] Ultrasonic disruption, purification of crude enzyme solution, etc. can be carried out according to conventional techniques in the art.

[0031] As disclosed in WO 2006 / 069799 A1, the (S)-2-chlorophenylalanine compound formed by the process of the present invention can be used to prepare optically active (S)-indoline-2-carboxylic acids, which in turn are intermediates in the production of active pharmaceutical ingredients, especially perindopril or its pharmaceutically acceptable salts.

[0032] Therefore, the present invention also relates to a method comprising preparing a (S)-2-chlorophenylalanine compound according to the aforementioned method of the present invention, and further converting the compound into perindopril or a pharmaceutically acceptable salt thereof. A method for converting (S)-2-chlorophenylalanine into (S)-indoline-2-carboxylic acid, and then further converting the (S)-indoline-2-carboxylic acid into perindopril or a salt thereof, can be referred to the prior art, such as the methods disclosed in WO2006 / 069799 and US4914214.

[0033] The phenylalanine ammonia-lyase mutant provided by the present invention can catalyze high-concentration cinnamic acid compounds to prepare L-phenylalanine compounds, thereby solving the problem of substrate inhibition in the catalytic reaction, thereby reducing production costs and meeting industrialization needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG1 is a diagram showing the SDS-PAGE results described in Example 4. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below with reference to specific examples, but the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to the different requirements of specific use, which are all considered to be within the scope of the present invention.

[0036] In the following examples, reagents and instruments not explicitly described are all conventional reagents and instruments in the art, can be obtained commercially, and used according to conventional methods in the art. Experimental procedures not explicitly described are all routine operations in the art; those skilled in the art will undoubtedly know how to perform the experiments and obtain the corresponding results based on the contents of the examples.

[0037] Example 1: Construction of wild-type phenylalanine ammonia lyase genetically engineered bacteria E. coli BL21 (DE3) / PAL

[0038] According to the amino acid sequence of PAL from Rhodotorula mucilaginosa in Genbank (Genbank accession number: P10248.2, as shown in SEQ ID NO: 2), after codon optimization (nucleotide sequence as shown in SEQ ID NO: 1), whole gene synthesis was performed and transferred into the vector pET28a(+) to obtain the pET28a(+)-PAL plasmid, which was then transformed into E. coli BL21(DE3) to obtain the wild-type phenylalanine ammonia lyase genetically engineered bacterium E. coli BL21(DE3) / PAL.

[0039] Example 2: Construction of recombinant phenylalanine ammonia lyase mutant genetically engineered bacteria by site-directed mutagenesis

[0040] N272K

[0041] Upstream primer 1: 5'-GGCCGTGCCcTTGACGAGACCGAGAC-3' (SEQ ID NO: 3)

[0042] Downstream primer 2: 5'-GTCTCGGTCTCGTCAAgGGCACGGCC-3' (SEQ ID NO: 4)

[0043] Y365F

[0044] Upstream primer 1: 5'-GGAGCGGGaACCGGTCCTGCCTGA-3' (SEQ ID NO: 5)

[0045] Downstream primer 2: 5'-TCAGGCAGGACCGGTtCCCGCTCC-3' (SEQ ID NO: 6)

[0046] The site-directed mutagenesis primers are as described above, with the mutation sites indicated in lowercase letters. Similarly, mutations were introduced by PCR using plasmid DNA containing the wild-type PAL gene (pET28a(+)-PAL) as a template.

[0047] The PCR reaction procedure was as follows: (1) pre-denaturation at 95°C for 3 min; (2) denaturation at 95°C for 30 s; (3) annealing at 60°C for 1 min; (4) extension at 68°C for 16 min; steps (2) to (4) were repeated for 16 cycles; (5) extension at 72°C for 10 min, followed by incubation at 4°C. The site-directed mutagenesis PCR product was digested with Kpn I at 37°C for 1 h.

[0048] The site-directed mutagenesis PCR products were transformed into E. coli DH5α competent cells, positive clones were picked for expansion and plasmid extraction, and mutant plasmid samples were sent to Biotechnology for sequencing verification of the correctness of the mutation site. After sequencing verification, the mutated plasmids were transformed into host bacteria Escherichia coli BL21(DE3) competent cells. Positive transformants were picked and sequenced to obtain genetically engineered bacteria E. coli BL21(DE3) / PAL-N272K, E. coli BL21(DE3) / PAL-Y365F, and E. coli BL21(DE3) / PAL-N272K / Y365F that can be inducibly expressed recombinant phenylalanine ammonia lyase; the corresponding PAL mutants were designated as mutants PAL-K (SEQ ID NO: 7), PAL-F (SEQ ID NO: 8), and PAL-KF (SEQ ID NO: 9), respectively.

[0049] Example 3: Preparation of wet cells of genetically engineered bacteria producing a recombinant phenylalanine ammonia-lyase mutant

[0050] The genetically engineered bacteria E. coli BL21 (DE3) / PAL-N272K, E. coli BL21 (DE3) / PAL-Y365F, and E. coli BL21 (DE3) / PAL-N272K / Y365F obtained in Example 2 were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance, respectively, and cultured at 37 ° C, 180 rpm for 10 h. Then, 2% (v / v) inoculum was inoculated into fresh TB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance, and cultured at 37 ° C, 180 rpm until the bacterial OD 600When the pH reaches 0.8-1.0, add IPTG to a final concentration of 0.1 mM. Induce the culture at 28°C for 12 hours, centrifuge at 6000 rpm for 10 minutes at 4°C, discard the supernatant, and collect the precipitate to obtain recombinant E. coli wet cells expressing the recombinant phenylalanine ammonia lyase mutant gene. This wet cell can be used directly as a biocatalyst or for protein purification. A similar method was used to prepare recombinant E. coli BL21(DE3) / PAL wet cells expressing the wild-type phenylalanine ammonia lyase gene.

[0051] Example 4: Isolation and purification of recombinant phenylalanine ammonia lyase and its mutants

[0052] Preparation of crude recombinant phenylalanine ammonia lyase enzyme solution: 0.2 g of the wet cells prepared in Example 3 were added to 9.8 mL of 100 mM sodium phosphate buffer solution, pH 8.0, and resuspended. The cells were ultrasonically disrupted under ice conditions to obtain a cell lysis solution. The solution was centrifuged at 6000 rpm and 4°C for 10 min to separate the precipitate and supernatant. The supernatant obtained was the desired crude enzyme solution.

[0053] The above precipitate was completely dissolved with an equal amount of 8 mol / L urea to obtain a precipitate solution.

[0054] 8 μL of each supernatant and precipitate solution were mixed with 2 μL of 5× loading buffer and heated in a 95°C metal bath for 10 minutes. The treated samples were then loaded onto a protein gel for electrophoresis. After the run, the gel was stained and destained, and an SDS-PAGE image was obtained using a protein gel imager.

[0055] Taking the preparation of crude enzyme solution from E. coli BL21(DE3) / PAL-N272K / Y365F wet cells prepared in Example 3 as an example, the resulting SDS-PAGE image is shown in FIG1 , wherein lane M is a protein marker; lane 1 from the left is the expression supernatant of recombinant phenylalanine ammonia lyase, and the boxed area is the target phenylalanine ammonia lyase protein; lane 2 from the left is the expression precipitate and lysate of the recombinant phenylalanine ammonia lyase; and lane 3 from the left is a blank expression.

[0056] Example 5: Determination of the conversion rate of recombinant phenylalanine ammonia lyase wild type and its mutants

[0057] The wet cells of the wild-type phenylalanine ammonia lyase PAL and the recombinant mutants PAL-K, PAL-F, and PAL-KF obtained in Example 3 were used to catalyze the substrate cinnamic acid.

[0058] The enzyme catalytic system composition and catalytic conditions are as follows: Prepare an amino donor solution, add trans-2-chloro-cinnamic acid, and dose to volume in a feed bottle; prepare an amino donor solution, add whole cells of a wild-type or mutant phenylalanine ammonia lyase strain, dose to volume in a reactor, and seal the reactor for uniform mixing. Continue adding the substrate solution until addition is complete and continue the conversion for 2 hours to obtain a conversion solution containing (S)-2-chlorophenylalanine. Conversion and chirality are determined by HPLC.

[0059] Chiral detection method: The chirality of phenylalanine ammonia lyase was determined by high performance liquid chromatography (HPLC). The chromatographic instrument was a high performance liquid chromatograph equipped with a UV detector; the chromatographic column was a CROWNPAK CR(+)150*4.0 mm, 5 μm column; the mobile phase was a 0.1% aqueous perchloric acid solution; the column temperature was 25°C, the detection wavelength was 210 nm, the flow rate was 0.8 mL / min, the injection volume was 5 μL, and the run time was 40 min.

[0060] Preferably, the amino donor is ammonia (NH3) or ammonia water (NH4OH), preferably ammonia water, which can also be ammonium hydroxide or ammonia monohydrate (NH3.H2O).

[0061] As a further preference, the amino donor is ammonia water, and the concentration of ammonia water can range from 3 to 6M, and is optimally 3 to 4M.

[0062] Preferably, the weight-to-volume ratio of the trans-2-chloro-cinnamic acid to the amino donor solution is 20-50 g / L, and optimally 40-50 g / L.

[0063] Preferably, the weight ratio of trans-2-chloro-cinnamic acid to wet bacteria is 0.5-4, preferably 1-3:1, and most preferably 2:1.

[0064] The product conversion rate was determined by high-performance liquid chromatography (HPLC). The chromatographic instrument was a high-performance liquid chromatograph equipped with a UV detector; the chromatographic column was a Waters symmetry C18 250 mm*4.6 mm, 5 μm column; the mobile phase was a 10 mM potassium dihydrogen phosphate solution (0.68 g potassium dihydrogen phosphate dissolved in 500 mL of water) in the aqueous phase A and methanol in the organic phase B; the column temperature was 25°C, the detection wavelength was 210 nm, the flow rate was 1.0 mL / min, and the injection volume was 20 μL.

[0065] Table 1: HPLC mobile phase gradient

[0066] The conversion rate was determined by area normalization (the sum of the contents of all the components in the peaks was taken as 100%, and the percentage of the area of ​​each peak to the total peak area was calculated).

[0067] Example 6: Use of wild-type phenylalanine ammonia lyase in the preparation of (S)-2-chlorophenylalanine

[0068] The recombinant Escherichia coli BL21 (DE3) / PAL wet bacteria containing the wild-type phenylalanine ammonia lyase gene obtained in Example 3 were used as biocatalysts to catalyze the substrate trans-2-chloro-cinnamic acid. The catalytic system and operation were as follows: 4M ammonia water was prepared, 150g of trans-2-chloro-cinnamic acid was added, the volume was adjusted to 1.5L, and the mixture was placed in a 2L feeding bottle; 4M ammonia water and 75g of phenylalanine ammonia lyase strain wet bacteria were prepared, the volume was adjusted to 1.5L, and the mixture was added to a 5L reactor. The reactor was sealed and stirred at 35°C and 400rpm. The substrate liquid was added at 200mL / h, and the addition was completed after 7.5h. The conversion was continued for 5h to obtain a conversion solution containing (S)-2-chlorophenylalanine. The conversion rate was 61.64% and the ee value was 99.8% by HPLC.

[0069] Example 7: Application of the phenylalanine ammonia lyase mutant PAL-K in the preparation of (S)-2-chlorophenylalanine

[0070] The wet cells expressing the phenylalanine ammonia lyase mutant PAL-K obtained in Example 3 were used as biocatalysts to catalyze the substrate trans-2-chloro-cinnamic acid. The catalytic system and operation were as follows: 4M ammonia water was prepared, 150g of trans-2-chloro-cinnamic acid was added, the volume was adjusted to 1.5L, and the mixture was placed in a 2L feeding bottle; 4M ammonia water was prepared, 75g of the wet cells of the phenylalanine ammonia lyase mutant strain was adjusted to 1.5L, and the mixture was added to a 5L reactor. The reactor was sealed and stirred at 35°C and 400rpm. The substrate liquid was added at 200mL / h, and the addition was completed after 7.5h. The conversion was continued for 5h to obtain a conversion solution containing (S)-2-chlorophenylalanine. The conversion rate was 81.9% and the ee value was 99.7% by HPLC.

[0071] Example 8: Application of the phenylalanine ammonia lyase mutant PAL-F in the preparation of (S)-2-chlorophenylalanine

[0072] The wet cells expressing the phenylalanine ammonia lyase mutant PAL-F obtained in Example 3 were used as biocatalysts to catalyze the substrate trans-2-chloro-cinnamic acid. The catalytic system and operation were as follows: 4M ammonia water was prepared, 150g of trans-2-chloro-cinnamic acid was added, the volume was adjusted to 1.5L, and the mixture was placed in a 2L feeding bottle; 4M ammonia water was prepared, 75g of the wet cells of the phenylalanine ammonia lyase mutant strain was adjusted to 1.5L, and the mixture was added to a 5L reactor. The reactor was sealed and stirred at 35°C and 400rpm. The substrate liquid was added at 200mL / h, and the addition was completed after 7.5h. The conversion was continued for 5h to obtain a conversion solution containing (S)-2-chlorophenylalanine. The conversion rate was 83.5% and the ee value was 99.8% by HPLC.

[0073] Example 9: Application of the phenylalanine ammonia lyase mutant PAL-KF in the preparation of (S)-2-chlorophenylalanine

[0074] The wet cells expressing the phenylalanine ammonia lyase mutant PAL-KF obtained in Example 3 were used as biocatalysts to catalyze the substrate trans-2-chloro-cinnamic acid. The catalytic system and operation were as follows: 4M ammonia water was prepared, 150g of trans-2-chloro-cinnamic acid was added, the volume was adjusted to 1.5L, and a 2L feeding bottle was placed. 4M ammonia water was prepared, 75g of the phenylalanine ammonia lyase mutant strain wet cells were adjusted to 1.5L, and added to a 5L reactor. The reactor was sealed and stirred at 35°C and 400rpm. The substrate liquid was added at 200mL / h, and the addition was completed after 7.5h. The conversion was continued for 5h to obtain a conversion solution containing (S)-2-chlorophenylalanine. The conversion rate was 89.08% and the ee value was 99.9% by HPLC.

[0075] Example 10: Application of the phenylalanine ammonia lyase mutant PAL-KF in the preparation of (S)-2-chlorophenylalanine

[0076] The wet cells expressing the phenylalanine ammonia lyase mutant PAL-KF obtained in Example 3 were used as biocatalysts to catalyze the substrate trans-2-chloro-cinnamic acid. The catalytic system and operation were as follows: 4M ammonia water was prepared, 150g of trans-2-chloro-cinnamic acid was added, the volume was adjusted to 1.5L, and the mixture was placed in a 2L feeding bottle; 4M ammonia water was prepared, 100g of the wet cells of the phenylalanine ammonia lyase mutant strain was adjusted to 1.5L, and the mixture was added to a 5L reactor. The reactor was sealed and stirred at 35°C and 400rpm. The substrate liquid was added at 200mL / h, and the addition was completed after 7.5h. The conversion was continued for 5h to obtain a conversion solution containing (S)-2-chlorophenylalanine. The conversion rate was 90.18% and the ee value was 99.8% by HPLC.

[0077] Example 11: Application of the phenylalanine ammonia lyase mutant PAL-KF in the preparation of (S)-2-chlorophenylalanine

[0078] The wet cells expressing the phenylalanine ammonia lyase mutant PAL-KF obtained in Example 3 were used as biocatalysts to catalyze the substrate trans-2-chloro-cinnamic acid. The catalytic system and operation were as follows: 4M ammonia water was prepared, 150g of trans-2-chloro-cinnamic acid was added, the volume was adjusted to 1.5L, and the mixture was placed in a 2L feeding bottle; 4M ammonia water was prepared, 50g of the wet cells of the phenylalanine ammonia lyase mutant strain was adjusted to 1.5L, and the mixture was added to a 5L reactor. The reactor was sealed and stirred at 35°C and 400rpm. The substrate liquid was added at 125mL / h, and the addition was completed after 12h. The conversion was continued for 5h to obtain a conversion solution containing (S)-2-chlorophenylalanine. The conversion rate was 88.93% and the ee value was 99.9% by HPLC.

[0079] Table 2: Conversion efficiency and chirality of wild-type and mutant phenylalanine ammonia lyase

[0080] sequence:

[0081] SEQ ID NO:1 nucleotide sequence after wild type optimization

[0082] SEQ ID NO: 2 Wild-type amino acid sequence

[0083] SEQ ID NO:7 PAL-K amino acid sequence

[0084] SEQ ID NO:8 PAL-F amino acid sequence

[0085] SEQ ID NO:9 PAL-KF amino acid sequence

Claims

1. A phenylalanine ammonia lyase mutant, wherein the asparagine at position 272 of the sequence shown in SEQ ID NO: 2 is mutated to lysine and / or the tyrosine at position 365 is mutated to phenylalanine; preferably, the amino acid sequence of the phenylalanine ammonia lyase mutant is shown in SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO:

9.

2. A nucleic acid molecule encoding the phenylalanine ammonia lyase mutant according to claim 1.

3. A recombinant vector comprising the nucleic acid molecule according to claim 2; Preferably, the starting vector of the recombinant vector can be a replication vector or an expression vector, and the expression vector can be a pET series vector, a shuttle vector, a phage or a viral vector; further preferably, the starting vector of the recombinant vector is pET28a(+).

4. A recombinant cell comprising the nucleic acid molecule of claim 2 or the recombinant vector of claim 3; preferably, the starting cell of the recombinant cell can be any cell capable of expressing a protein; further preferably, the recombinant cell is a genetically engineered bacterium, and its starting bacterium is E. coli BL21 (DE3). A composition comprising the phenylalanine ammonia lyase mutant according to claim 1 .

6. Use of the phenylalanine ammonia-lyase mutant according to claim 1, the nucleic acid molecule according to claim 2, the recombinant vector according to claim 3, the recombinant cell according to claim 4, or the composition according to claim 5 in synthesizing L-phenylalanine compounds; preferably, the L-phenylalanine compound is (S)-2-chlorophenylalanine.

7. A method for synthesizing L-phenylalanine compounds, comprising the following steps: The phenylalanine ammonia lyase mutant according to claim 1 or the fermentation culture of the recombinant cell according to claim 4 or the composition according to claim 5 is added to a reaction system containing a substrate and an amino donor solution for reaction, wherein the L-phenylalanine compound is (S)-2-chlorophenylalanine, the substrate is trans-2-chloro-cinnamic acid, and the amino donor solution is ammonia (NH3) or an ammonia solution; preferably, the fermentation culture of the recombinant cell can be a wet cell obtained by fermentation culture, or a crude enzyme solution extracted after ultrasonic disruption of the wet cell, or a purified pure enzyme solution.

8. The method according to claim 7, wherein: The amino donor solution is ammonia water, and the concentration of ammonia water ranges from 3 to 6 M, preferably from 3 to 4 M; The weight-to-volume ratio of the trans-2-chloro-cinnamic acid to the amino donor solution is 20-50 g / L, preferably 40-50 g / L; The weight ratio of the trans-2-chloro-cinnamic acid to the wet bacteria is 0.5 to 4:1, preferably 1 to 3:1, and most preferably 2:

1.

9. The method according to claim 7 or 8, wherein The method comprises the following steps: mixing wet cells obtained by fermentation culture of the recombinant cell according to claim 4, or crude enzyme solution extracted after ultrasonic crushing of the wet cells, or purified pure enzyme solution with half amount of ammonia water, placing the mixture into a reactor for reaction at 30° C., mixing the other half amount of ammonia water and trans-2-chloro-cinnamic acid to prepare a supplementary feed, adding the feed at a rate of 125 mL to 200 mL / h, and continuing the conversion for 5 hours after the addition is completed, thereby obtaining the product.

10. A method for producing perindopril or a pharmaceutically acceptable salt thereof, comprising preparing (S)-2-chlorophenylalanine by the method according to any one of claims 7 to 9, converting the (S)-2-chlorophenylalanine into (S)-indoline-2-carboxylic acid, and further converting the (S)-indoline-2-carboxylic acid into perindopril or a salt thereof.