Phenylalanine ammonialyase and application thereof

Abstract

The invention discloses phenylalanine ammonialyase, the amino acid sequence of the phenylalanine ammonialyase is SEQ ID NO: 3 or SEQ ID NO: 5, and the phenylalanine ammonialyase is obtained by site-directed mutagenesis on the basis of the phenylalanine ammonialyase from a microorganism Photorabus lamondii subsp.lamondii. Compared with phenylalanine ammonialyase from other sources, the phenylalanine ammonialyase mutant provided by the invention can catalyze a high-concentration substrate, has higher catalytic activity, wider substrate range and higher yield, is lower in production cost, and can be used for industrial production of 2-chlorophenylalanine.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biocatalysis, and in particular, relates to a phenylalanine ammonia-lyase and its application, especially in the synthesis of 2-chlorophenylalanine. BACKGROUND

[0002] 2-chlorophenylalanine is a halogenated version of the amino acid phenylalanine and is a precursor to several neurotransmitters including dopamine, adrenaline and noradrenaline as well as melanin and tyrosine.

[0003] 2-chlorophenylalanine is an amino acid derivative that is widely used in scientific research. It is also an over-the-counter medication commonly used to relieve pain, reduce fever and inflammation, and is found in some cold medications. It can be used as an intermediate in organic synthesis to synthesize other compounds, including pesticides and pharmaceuticals. Researchers use 2-chlorophenylalanine to detect the complexity of protein, enzyme, nucleic acid structure and function. It also helps to explore the effects of different drugs and toxins on cellular processes. In addition, 2-chlorophenylalanine plays an important role in studying the effects of environmental pollutants on the human body.

[0004] WO2006 / 069799A1 discloses a process for the preparation of enantiomerically enriched phenylalanine compounds, in one of the embodiments, (S)-2-chloro-phenylalanine is prepared by reacting 2-Cl-cinnamic acid with ammonia using a phenylalanine ammonia-lyase derived from Rhodotorula glutinis. The disadvantage of this process is that strong substrate and product inhibition occurs, thus requiring the process to be carried out at low concentrations. In case of a demand for large scale production of the product, either larger volumes or more complex feeding schemes are required, which makes this process uncompetitive from an industrial production point of view.

[0005] CN101517089A discloses an improved process for the preparation of phenylalanine compounds, the phenylalanine ammonia-lyase used is derived from Idiomarina loihiensis and is used to catalyze the reaction of the corresponding cinnamic acid compound with an amino donor. This method effectively reduces substrate inhibition and has higher activity compared to the phenylalanine ammonia-lyase derived from R. glutinis. However, in actual production, the catalytic activity of the phenylalanine ammonia-lyase derived from I. loihiensis is still low, which cannot meet the requirements of industrial production.

[0006] CN106755160A discloses an improved process for preparing phenylalanine compounds, the source of histidine ammonia lyase used is Pseudoalteromonas sp. P1-26, which is used to produce L-phenylalanine compounds. Compared with the above-mentioned process, the method can reduce production cost and improve production efficiency. Although the yield is improved compared with the two methods described above, the amount of enzyme required in production is too high, which is not conducive to large-scale promotion.

[0007] CN116949024A discloses a phenylalanine ammonia lyase mutant and its application in the synthesis of (S)-2-chloro-phenylalanine. The mutant has higher catalytic activity and wider substrate range, which can improve the conversion efficiency and has high application prospect. However, the wild enzyme of the mutant is derived from microorganism Idiomarina abyssalis, which belongs to the same genus as CN101517089A. In order to enrich the high-efficiency catalytic enzyme in the synthesis of 2-chloro-phenylalanine, it is still necessary to screen phenylalanine ammonia lyases from different sources. SUMMARY

[0008] In order to develop a method capable of catalyzing 2-chloro-phenylalanine to produce 2-chloro-phenylalanine by one-step enzyme reaction, the inventors have screened a large number of amino acid ammonia lyases, and unexpectedly found that a phenylalanine ammonia lyase (Uniport ID accession number Q7N4T3, named PAL4 herein) derived from Photorhabdus laumondii subsp. laumondii can catalyze 2-chloro-phenylalanine to produce 2-chloro-phenylalanine in the presence of an amino donor. Based on the enzyme, the inventors obtained two phenylalanine ammonia lyase mutants with industrial application prospect through codon optimization and further directed evolution.

[0009] Specifically, the present application comprises the following technical solutions.

[0010] The first object of the present application is to provide a phenylalanine ammonia lyase, the amino acid sequence of which is:

[0011] SEQ ID NO: 3, which is a mutant of SEQ ID NO: 1 with L replaced by I at position 199; or

[0012] SEQ ID NO: 5, which is a mutant of SEQ ID NO: 1 with H replaced by M at position 133 and L replaced by I at position 199.

[0013] SEQ ID NO: 3 is:

[0014] MKAKDVQPTIIINKNGLISLEDIYDIAIKQKKVEISTEITELLTHGREKLEEKLNSGEVIY GINTGFGGNANLVVPFEKIAEHQQNLLTFLSAGTGDYMSKPCIKASQFTMLLSVCKGWSA TRPIVAQAIVDHINHDIVPLVPRYGSVGASGDLIPLSYIARALCGIGKVYYMGAEIDAAE AIKRAGLTPLSLKAKEGIALINGTRVMSGISAITVIKLEKLFKASISAIALAVEALLASHE HYDARIQQVKNHPGQNAVASALRNLLAGSTQVNLLSGVKEQANKACRHQEITQLNDTLQ EVYSIRCAPQVLGIVPESLATARKILEREVISANDNPLIDPENGDVLHGGNFMGQYVART MDALKLDIALIANHLHAIVALMMDNRFSRGLPNSLSPTPGMYQGFKGVQLSQTALVAAI RHDCAASGIHTLATEQYNQDIVSLGLHAAQDVLEMEQKLRNIVSMTILVVCQAIHLRGNI SEIAPETAKFYHAVREISSPLITDRALDEDIIRIADAIINDQLPLPEIMLEE.

[0015] wherein the sequence of SEQ ID NO: 5 is:

[0016] MKAKDVQPTIIINKNGLISLEDIYDIAIKQKKVEISTEITELLTHGREKLEEKLNSGEVIY GINTGFGGNANLVVPFEKIAEHQQNLLTFLSAGTGDYMSKPCIKASQFTMLLSVCKGWSA TRPIVAQAIVDMINHDIVPLVPRYGSVGASGDLIPLSYIARALCGIGKVYYMGAEIDAAE AIKRAGLTPLSLKAKEGIALINGTRVMSGISAITVIKLEKLFKASISAIALAVEALLASHEH YDARIQQVKNHPGQNAVASALRNLLAGSTQVNLLSGVKEQANKACRHQEITQLNDTLQ EVYSIRCAPQVLGIVPESLATARKILEREVISANDNPLIDPENGDVLHGGNFMGQYVART MDALKLDIALIANHLHAIVALMMDNRFSRGLPNSLSPTPGMYQGFKGVQLSQTALVAAI RHDCAASGIHTLATEQYNQDIVSLGLHAAQDVLEMEQKLRNIVSMTILVVCQAIHLRGNI SEIAPETAKFYHAVREISSPLITDRALDEDIIRIADAIINDQLPLPEIMLEE.

[0017] A second object of the present application is to provide a gene encoding the phenylalanine ammonia-lyase described above.

[0018] Preferably, the gene encoding the phenylalanine ammonia-lyase described above has a nucleotide sequence as shown in SEQ ID NO. 4 or SEQ ID NO. 6.

[0019] A third object of the present application is to provide an expression vector comprising the gene described above.

[0020] In one or more embodiments, the expression vector is a plasmid. Preferably, the vector is selected from a pET-28a(+) plasmid, a pET-28b(+) plasmid, or a pET-20b(+) plasmid.

[0021] A fourth object of the present application is to provide a host cell comprising the phenylalanine ammonia-lyase described above.

[0022] In one or more embodiments, the host cell is a microorganism. Preferably, the host cell is selected from Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica, Bacillus subtilis, and the like.

[0023] The fifth aspect of the present application aims to provide the phenylalanine ammonia-lyase or the host cell described above for use in the enzymatic synthesis of 2-chlorophenylalanine.

[0024] The sixth aspect of the present application aims to provide a method for preparing 2-chlorophenylalanine, which comprises the step of adding the phenylalanine ammonia-lyase described above or an expression system containing the enzyme to a reaction system containing the substrate 2-chlorophenylacrylic acid to perform a reaction.

[0025] In one or more embodiments, the method for preparing further comprises the step of adding an amino donor to the reaction system, the amino donor being ammonia or aqueous ammonia.

[0026] In one or more embodiments, the reaction temperature in the method for preparing is 30-50℃, and the reaction pH value is 9.0-11.

[0027] The present application is based on the wild-type phenylalanine ammonia-lyase from the microorganism Photorhabdus laumondii subsp. laumondii, and the phenylalanine ammonia-lyase mutant with significantly increased catalytic activity is finally obtained by modifying the molecular structure of the phenylalanine ammonia-lyase through site-directed mutagenesis technology. Compared with other sources of phenylalanine ammonia-lyase, the phenylalanine ammonia-lyase mutant provided by the present application can catalyze high-concentration substrates, has higher catalytic activity, a wider substrate range and higher yield, has lower production cost, and can be used for industrial production of 2-chlorophenylalanine. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is the structural diagram of the plasmid pET28a-PAL4 constructed by the present application for expressing the wild-type enzyme PAL4.

[0029] Figure 2 is the HPLC detection diagram in the reaction process of the wild-type phenylalanine ammonia-lyase catalyzing 2-chlorophenylacrylic acid to generate 2-chlorophenylalanine.

[0030] Figure 3 is the HPLC detection diagram in the reaction process of the phenylalanine ammonia-lyase with the amino acid sequence of SEQ ID NO: 3 catalyzing 2-chlorophenylacrylic acid to generate 2-chlorophenylalanine. DETAILED DESCRIPTION

[0031] The present inventors have screened a large number of amino acid deaminases and unexpectedly found that a phenylalanine deaminase (Uniport ID No. Q7N4T3, designated as PAL4 herein) derived from Photorhabdus laumondii subsp. laumondii can catalyze 2-chlorophenylacrylic acid to generate 2-chlorophenylalanine in the presence of an amino donor. The amino acid sequence (SEQ ID NO: 1) of the wild-type PAL4 is as follows:

[0032] MKAKDVQPTIIINKNGLISLEDIYDIAIKQKKVEISTEITELLTHGREKLEEKLNSGEVIYGINTGFGGNANLVVPFEKIAEHQQNLLTFLSAGTGDYMSKPCIKASQFTMLLSVCKGWSATRPIVAQAIVDHINHDIVPLVPRYGSVGASGDLIPLSYIARALCGIGKVYYMGAEIDAAEAIKRAGLTPLSLKAKEGLALINGTRVMSGISAITVIKLEKLFKASISAIALAVEALLASHEHYDARIQQVKNHPGQNAVASALRNLLAGSTQVNLLSGVKEQANKACRHQEITQLNDTLQEVYSIRCAPQVLGIVPESLATARKILEREVISANDNPLIDPENGDVLHGGNFMGQYVARTMDALKLDIALIANHLHAIVALMMDNRFSRGLPNSLSPTPGMYQGFKGVQLSQTALVAAIRHDCAASGIHTLATEQYNQDIVSLGLHAAQDVLEMEQKLRNIVSMTILVVCQAIHLRGNISEIAPETAKFYHAVREISSPLITDRALDEDIIRIADAIINDQLPLPEIMLEE.

[0033] Although the wild-type PAL4 described above can catalyze 2-chlorophenylacrylic acid to generate 2-chlorophenylalanine, its catalytic activity is still low and cannot be used for industrial production. The inventors have rationally designed the amino acids in the range of -10 to +10 from the active site of the enzyme and the substrate binding site of the enzyme by molecular dynamics simulation to determine the mutation points with higher affinity of enzyme and substrate binding after mutation, and further screened two PAL4 mutants with significantly improved enzyme activity, L199I and L199I / H133M.

[0034] In the present text, the terms "wild-type", "wild-type (phenylalanine ammonia-lyase)", "wild-type PAL4" have the same meaning and refer to a phenylalanine ammonia-lyase PAL4 (Uniport ID entry Q7N4T3) having the amino acid sequence of SEQ ID NO: 1. Correspondingly, the terms "mutant", "mutant enzyme", "phenylalanine ammonia-lyase mutant" have the same meaning and refer to an enzyme which has been modified in its amino acid sequence with respect to the wild-type enzyme PAL4, while retaining the same catalytic reaction characteristics, in particular to an enzyme having an improved enzyme activity and / or an improved enzyme activity stability, such as the mutants having the amino acid sequences of SEQ ID NO: 3 and 5. For the sake of convenience, the wild-type phenylalanine ammonia-lyases and their mutants can be referred to as "phenylalanine ammonia-lyases" in the present text.

[0035] In the present text, the term "(enzyme activity) improvement" as used above means an improvement of at least 100% compared to a reference level, such as the enzyme activity of PAL4, such as at least 1-fold, at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 20-fold compared to the reference level.

[0036] It is to be understood that the term "mutation" as used herein includes, but is not limited to, substitution, deletion, insertion, chemical modification of an amino acid residue, preferably a mutation which is a positive mutation, i.e. an enzyme activity improving mutation. The substitution can be a non-conservative substitution, a conservative substitution or a combination of non-conservative and conservative substitutions. A "conservative" amino acid substitution or mutation refers to the interchangeability of residues having similar side chains and thus generally includes the substitution of an amino acid in a polypeptide with another amino acid of the same or similar class. However, as used herein, a conservative mutation does not include a substitution of hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxyl-containing to hydroxyl-containing, or small residue to small residue if the conservative mutation can alternatively be an aliphatic to aliphatic, non-polar to non-polar, polar to polar, acidic to acidic, basic to basic, aromatic to aromatic, or a restricted residue to restricted residue substitution. It is well known in the art that common instances of conservative substitutions include: interchanges between aromatic amino acids F, W, Y; interchanges between hydrophobic amino acids L, I, V; interchanges between polar amino acids Q, N; interchanges between basic amino acids K, R, H; interchanges between acidic amino acids D, E; interchanges between hydroxyl amino acids S, T. In addition, A, V, L or I can be conservatively mutated to another aliphatic residue or another non-polar residue. Exemplary conservative substitutions can be made, for example, according to the following table, wherein amino acids belonging to the same partition in the second column can be substituted for one another, and in preferred cases amino acids in the same row in the third column can be substituted for one another:

[0037]

[0038] A "non-conservative substitution" is a substitution or mutation of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. A non-conservative substitution can use amino acids from the groups listed above, but not within the group. In one embodiment, a non-conservative mutation affects (a) the structure of the peptide backbone in the region of the substitution (e.g., proline in place of glycine), (b) the charge or hydrophobicity, or (c) the bulk of the side chain.

[0039] A "deletion" refers to a modification of a polypeptide by removing one or more amino acids from a reference polypeptide. A deletion can include removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids comprising the reference enzyme, while retaining enzyme activity and / or retaining the improved properties of the engineered maleate hydratase. Deletions can be to the interior and / or the ends of the polypeptide. In embodiments, deletions can comprise contiguous segments or can be non-contiguous.

[0040] An "insertion" refers to a modification of a polypeptide by adding one or more amino acids to a reference polypeptide. In some embodiments, the improved engineered maleate hydratase includes one or more amino acid insertions into a naturally occurring maleate hydratase and one or more amino acid insertions into other improved maleate hydratase polypeptides. The insertion can be internal to the polypeptide, or carboxy terminal or amino terminal. Insertions as used herein include fusion proteins as known in the art. The insertion can be a contiguous segment of amino acids or separated by one or more amino acids in the naturally occurring polypeptide.

[0041] The amino acid sequences of the phenylalanine ammonia-lyase mutants of the present application are explicit, and thus the genes encoding them, expression cassettes and plasmids containing these genes, and transformants containing the plasmids are readily available to those skilled in the art.

[0042] In order to express the phenylalanine ammonia-lyase or its mutants most optimally in Escherichia coli, which is most commonly used in genetic engineering, the expression genes of these enzymes can be codon-optimized.

[0043] Codon optimization is a technique that can be used to maximize protein expression in an organism by increasing the efficiency of translation of a gene of interest. Different organisms typically show a particular bias for one of the codons that encodes the same amino acid due to mutational bias and natural selection. For example, in fast-growing microorganisms such as Escherichia coli, the optimized codons reflect the composition of their respective genomic tRNA pools. Thus, in fast-growing microorganisms, low-frequency codons for an amino acid can be replaced with a codon for the same amino acid but of high frequency. Thus, the expression of the optimized DNA sequence is improved in fast-growing microorganisms.

[0044] For example, for expressing phenylalanine ammonia-lyase in E. coli, the coding gene of the codon-optimized amino acid sequence SEQ ID NO: 1 can be SEQ ID NO: 2.

[0045] These genes, expression cassettes, plasmids, transformants can be obtained by genetic engineering methods well known to those skilled in the art.

[0046] The transformant host can be any microorganism suitable for expressing phenylalanine ammonia-lyase, including bacteria and fungi. The preferred microorganism is Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, or E. coli, preferably E. coli, more preferably E. coli BL21 (DE3).

[0047] When used as a biocatalyst, the phenylalanine ammonia-lyase of the present application can be added to the reaction system in the form of an enzyme or in the form of a bacterial cell. The enzyme form includes free enzyme, immobilized enzyme, including purified enzyme, crude enzyme, fermentation broth, carrier immobilized enzyme, etc.; the bacterial cell form includes viable bacterial cells, dead bacterial cells, immobilized bacterial cells, etc.

[0048] When microorganisms such as Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, or E. coli are no longer used for fermentation proliferation, but are used for enzyme catalysis, they are a natural immobilized enzyme by themselves, and do not need to be broken down, even extracted and purified, and can be directly used as an enzyme preparation for catalysis. Since the reaction substrate and the reaction product are both small molecule compounds, they can easily pass through the biological barrier of the bacterial cell membrane, so the bacterial cells do not need to be broken down, which is economically advantageous.

[0049] The present application is further described in detail below in conjunction with specific examples. It should be understood that the following examples are for illustration only and are not intended to limit the scope of the present application.

[0050] Embodiments

[0051] In this text, the addition amount, content and concentration of various substances are mentioned, and the percentage content mentioned herein refers to the mass percentage content, unless otherwise specified.

[0052] Materials and Methods

[0053] The whole gene synthesis, primer synthesis and sequencing in the examples were completed by Suzhou Jinyuizhi Biological Technology Co., Ltd.

[0054] The molecular biology experiments in the examples include plasmid construction, enzyme digestion, ligation, preparation of competent cells, transformation, medium preparation, etc., which are mainly performed according to the third edition of Molecular Cloning Guide (J. Sambrook, D. W. Russell (USA) edited, Huang Peitang et al. translated, Science Press, Beijing, 2002). If necessary, the specific experimental conditions can be determined by simple tests.

[0055] The PCR amplification experiments are performed according to the reaction conditions or kit instructions provided by the plasmid or DNA template supplier. If necessary, they can be adjusted by simple tests.

[0056] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2. (LB solid medium additionally adds 20 g / L agar powder.)

[0057] TB medium: 24 g / L yeast extract, 12 g / L tryptone, 16.43 g / L K2HPO4.3H2O, 2.31 g / L KH2PO4, 5 g / L glycerol, pH 7.0-7.5. (TB solid medium additionally adds 20 g / L agar powder.)

[0058] HPLC detection conditions of substrate 2-chlorophenylacrylic acid and product 2-chlorophenylalanine:

[0059] Agilent high performance liquid chromatograph 1260 (or the same type); chromatographic column: Agilent SB-Aq, 4.6 mm x 250 mm, 5 μm; mobile phase: water: methanol = 90:10; flow rate: 1.0 mL / min; column temperature: 25°C; wavelength: 210 nm, injection volume 5 μL.

[0060] It should be noted that for the sake of convenience of description, in the examples, the strain number, plasmid number, enzyme number, and enzyme-encoding gene number can share a number, which is easily understood by those skilled in the art, i.e., the same number can refer to different biological forms in different environments.

[0061] Example 1: Construction of wild-type enzyme PAL4 engineering strain

[0062] According to the amino acid sequence SEQ ID NO: 1 of phenylalanine ammonia lyase PAL4 (Uniport ID accession number Q7N4T3) derived from Photorhabdus laumondii subsp. laumondii, its encoding gene SEQ ID NO: 2 is designed by expressing optimization of E. coli preferred codons. Suzhou Jinyuzhi Biotechnology Co., Ltd. performs full gene sequence synthesis and cloning into plasmid pET28a, 3' end insertion into XhoI site, 5' end seamless cloning, to obtain plasmid pET28a-PAL4, seeFigure 1 .

[0063] The recombinant plasmid pET28a-PAL4 is transformed into an expression host such as E. coli BL21 (DE3) competent cells (Invitrogen Corporation) by electroporation to obtain a recombinant E. coli expressing wild-type phenylalanine ammonia-lyase PAL4.

[0064] The recombinant plasmid pET28a-PAL4 can also be transformed into other hosts such as Pichia pastoris, Bacillus subtilis, etc. to express wild-type phenylalanine ammonia-lyase PAL4.

[0065] Example 2: Construction of PAL4 mutant L199I engineering strain

[0066] 2.1 Construction of saturated mutant library

[0067] Molecular docking was performed on wild-type phenylalanine ammonia-lyase PAL4 and substrate (2-chlorophenylacrylic acid) to determine the binding of enzyme and substrate Amino acids within the range were rationally designed, and the mutation points with higher affinity of enzyme and substrate binding after mutation were determined by molecular dynamics simulation. We designed and selected 24 amino acids at positions 61, 62, 65, 67, 68, 87, 91, 92, 133, 151, 154, 167, 199, 202, 203, 337, 338, 353, 432, 433, 435, 438, 439, and 470 of phenylalanine ammonia-lyase PAL4 for saturation mutation.

[0068] Taking the mutation of L199 as an example, the PCR primers were designed as follows:

[0069] Forward primer PAL4-199-F: AGGCGAAGGAGGGCNNKGCATTAATCAACGGT (SEQ ID NO: 7),

[0070] Reverse primer PAL4-199-R: GCCCTCCTTCGCCTTCAGAGACAAT (SEQ ID NO: 8).

[0071] The 50 μL PCR reaction system comprises: 50 ng of plasmid template pET28a-PAL4, 30 pmol of a pair of primers PAL4-199-F and PAL4-199-R, 25 μL of PrimeSTAR Max Premix (2X), and the rest is supplemented with ddH2O.

[0072] PCR reaction conditions: 95°C for 5 min; 94°C for 30 s, 60°C for 30 s, 72°C for 30 s / kbp; 25 cycles; 72°C for 10 min. After PCR, 5 μL of the PCR product was taken for gel running confirmation. The correct band size was used to digest the plasmid template with Dpn I to obtain the mutant plasmid pET28a-PAL4-L199, which was then electroporated into E. coli BL21 (DE3) to obtain the saturated mutant library.

[0073] 2.2 Screening of mutants by enzyme activity

[0074] The wild-type PAL4 and the mutant strains were inoculated and induced, and the bacterial cells were collected for purification to prepare pure enzymes. The specific steps are as follows: the collected bacterial cells were resuspended with 20 mL of equilibration buffer (50 mM HEPES, 50 mM NaCl, 10 mM imidazole, pH 7.5) to obtain a bacterial suspension, which was subjected to ultrasonic disruption under ice bath conditions to obtain a crude enzyme solution. The crude enzyme solution was centrifuged at 12000 rpm for 30 min to collect the supernatant, which was used to extract the pure enzyme using a nickel column. First, the nickel column was rinsed with water, then 10 column volumes of equilibration buffer were used to rinse and equilibrate the nickel column. The supernatant was loaded and combined with the filler for a period of time. The impurities were eluted with 20 mL of wash buffer (50 mM HEPES, 50 mM NaCl, 25 mM imidazole, pH 7.5), and the target protein was eluted with 4 mL of elution buffer (50 mM HEPES, 50 mM NaCl, 250 mM imidazole, pH 7.5). Each 1 mL was aliquoted in a 1.5 mL EP tube, which was the pure enzyme solution.

[0075] The pure enzyme was used for catalytic reaction, and the conditions are as follows:

[0076]

[0077] The reaction volume was 1 mL, which was placed in a 40°C, 180 rpm shaker for 24 h. The sample was taken for liquid phase detection. The enzyme activity was determined by comparing the concentration of the product 2-chlorophenylalanine. The liquid phase detection results of the wild-type PAL4 and the mutant L199I are shown in Figure 2 and Figure 3 The specific liquid phase detection conditions are as follows:

[0078]

[0079] Most of the mutant strains with no improvement or even reduction in enzyme activity were discarded. The mutant strains with enzyme activity improved by more than 50% were selected, and the plasmid was extracted. Through gene sequencing and amino acid site comparison, it was found that the L199I mutation (the sequence is shown in SEQ ID NO: 3) could increase the enzyme activity of the wild-type enzyme PAL4 by more than 2 times, which belonged to the best result in the positive mutation. The specific results are shown in the table below:

[0080]

[0081]

[0082] Example 3: Construction of PAL4 mutant L199I / H133M engineering strain

[0083] On the basis of the above-mentioned mutant L199I, iterative saturation mutation was carried out, and the specific steps were as follows:

[0084] Taking L199I / H133M as an example, the primers were designed as follows:

[0085] Forward primer PAL4-199 / 133-F: CAAGCGATTGTCGACNNKATCAACCACGATATCGT (9 SEQ ID NO: 9),

[0086] Reverse primer PAL4-199 / 133-R: GTCGACAATCGCTTGAGCTACAATCGGG (SEQ ID NO: 10).

[0087] The 50 μL PCR reaction system included: 50 ng plasmid template pET28a-PAL4-L199I, 30 pmol of a pair of primers PAL4-199 / 133-F and PAL4-199 / 133-R, 25 μL PrimeSTAR Max Premix (2X), and the rest was supplemented with ddH2O.

[0088] The PCR reaction conditions were: 95°C for 5 min; 94°C for 30 s, 60°C for 30 s, 72°C for 30 s / kbp; 25 cycles; 72°C for 10 min. After the PCR was completed, 5 μL of the PCR product was taken for gel running confirmation, and the band size was correct. Then, DpnI was used to digest the plasmid template, and then E. coli BL21 (DE3) was electrotransformed to obtain a saturation mutant library.

[0089] According to the steps of Example 2, the mutants were screened, and most of the mutants with no improvement or even reduction in enzyme activity were discarded; the mutant strains with improved enzyme activity relative to the mutant L199I were selected, the plasmid was extracted, and gene sequencing and amino acid site comparison were performed, and it was found that the L199I / H133M (the sequence is shown as SEQ ID NO: 5) mutation could further improve the enzyme activity of the wild-type enzyme PAL4, which belonged to the best result in the positive mutation, and the specific results are shown in the following table:

[0090]

[0091] Example 4: Fermentation and catalytic reaction of engineering strain

[0092] The wild type and two mutant strains were subjected to shake flask fermentation, the strains were inoculated into 5 mL LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C, 220 rpm overnight for 12 h, inoculated into LB liquid medium containing 50 μg / mL kanamycin at 1% v / v inoculation amount, and cultured at 37°C until OD 600 When OD was 0.6-0.8, 0.5 mmol / L IPTG was added, and the expression was induced at 18°C, 220 rpm for 12 h, then the bacteria were collected by centrifugation at 4°C, 4000 rpm for 10 min.

[0093] According to the steps in Example 2, the bacteria were used for purification to prepare pure enzyme, and then catalytic reaction was carried out under the condition of high concentration of 2-chlorophenylacrylic acid (1M), and the reaction conditions were as follows:

[0094]

[0095] The pH was adjusted to 10.0 by hydrochloric acid, and the reaction was carried out at 40°C, 250 rpm overnight, and the sample was subjected to liquid phase detection, and the results were as follows:

[0096]

[0097] The above results show that the two mutant strains can efficiently catalyze 2-chlorophenylacrylic acid to generate 2-chlorophenylalanine whether in the presence of low concentration or high concentration of substrate, and can be used for industrial production.

[0098] The above is only the preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A phenylalanine ammonia-lyase, the amino acid sequence of which is: SEQ ID NO:3 is a mutant of SEQ ID NO:1 where the L at position 199 is replaced with I; or SEQ ID NO:5 is a mutant of SEQ ID NO:1 where H at position 133 is replaced with M and L at position 199 is replaced with I.

2. The gene encoding the phenylalanine ammonia-lyase of claim 1.

3. The gene as described in claim 2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.4 or SEQ ID NO.

6.

4. An expression vector comprising the gene of claim 2 or 3.

5. The expression vector as described in claim 4, characterized in that, The vector is selected from pET-28a(+) plasmid, pET-28b(+) plasmid or pET-20b(+) plasmid.

6. A host cell comprising the phenylalanine ammonia-lyase of claim 1.

7. The host cell as described in claim 6, characterized in that, The host cells are selected from Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Yersinia lipolytica, and Bacillus subtilis.

8. The use of the phenylalanine ammonia-lyase of claim 1 and the host cell of claim 6 or 7 in the enzymatic synthesis of 2-chlorophenylalanine.

9. A method for preparing 2-chlorophenylalanine, characterized in that, The method includes the step of adding the phenylalanine ammonia-lyase of claim 1 or an expression system containing the enzyme to a reaction system containing the substrate 2-chlorophenylacrylic acid for reaction.

10. The preparation method according to claim 9, characterized in that, The method further includes the step of adding an amino donor to the reaction system, wherein the amino donor is ammonia or ammonia water.

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