Phenylalanine aminolyase mutant and its application

Abstract

The present invention belongs to the field of biomedicine technology, and is particularly related to phenylalanine aminolyase mutants and applications thereof. Specifically, the present invention provides the application of phenylalanine ammonia lyase mutants as catalysts for catalyzing substrate 9-anthracene acrylic acid to generate 3-(9-anthryl) alanine, wherein the phenylalanine ammonia lyase mutant is a variant obtained by mutating the amino acid sequence of phenylalanine ammonia lyase E1-101-2-3 based on EU numbering amino acid substitution site F137G or L138V. The present invention obtains a phenylalanine aminolyase mutant with high specificity and high catalytic activity by enzyme mutation and screening, and the mutant is applied in the field of non-natural amino acid synthesis to efficiently catalyze 9-anthracene acrylic acid to generate 3-(9-anthryl) alanine.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a phenylalanine aminolyase mutant and an application thereof. Background Art

[0002] The human body contains 20 natural amino acids. The number of functional groups carried by natural amino acids is limited, which cannot meet people's needs for protein structure and function in biological science research and applications. Non-natural amino acids (nAAs) can be site-specifically introduced into target proteins with little disturbance to the protein structure. Moreover, non-natural amino acids can achieve functional diversification of modified proteins by replacing the side chain groups of amino acids. At present, non-natural amino acids are widely present in biologically active natural products, peptide drugs and functional non-natural proteins, and they perform well in protein modification in bacteria, mammalian cells, etc., and have broad development space. How to efficiently and stereoselectively construct non-natural amino acids has become the main goal of synthetic chemistry and synthetic biology.

[0003] Unlike natural amino acids, non-natural amino acids cannot be synthesized autonomously during microbial metabolism, or the synthesis efficiency of non-natural amino acids is very low. Therefore, if non-natural amino acids are to be synthesized, it is usually necessary to reconstruct the synthesis pathway of the target product or add specific enzymes. How to screen enzyme mutants that can catalyze the synthesis of non-natural amino acids is a problem of great concern to modern enzyme engineering technicians.

[0004] Phenylalanine amino-lyase (PAL), also known as phenylalanine ammonia-lyase, is an acidic protein and an intracellular induced enzyme. PAL protein has four subunits. The molecular weight of each subunit is between 55 and 88 KD. The four subunits form an oligomer. The molecular weight of PAL protein is usually 220~330 KDa. The molecular weights of different types of PAL are slightly different. For example, the molecular weight of PAL in wheat is 280 KDa, the molecular weight of PAL in corn is 306 KDa, and the molecular weight of PAL in rice is 280 KDa. PAL has an electrophilic center of dehydroalanyl in the enzyme active site. Moreover, phenylalanine ammonia-lyase is a type of aromatic amino acid ammonia-lyase. Under natural conditions, aromatic amino acid ammonia-lyase undergoes a deamination reaction to hydrolyze the amino group of aromatic amino acids, and a reversible reaction can also occur under certain conditions. Therefore, based on the above reversible reaction, acids can be biosynthesized into non-natural amino acids using phenylalanine ammonia-lyase.

[0005] Enzyme mutants controllably cut, modify or mutate natural enzyme genes, thereby changing the catalytic properties, substrate specificity or coenzyme specificity of these enzymes to make them more in line with various needs. In the current prior art, the efficiency of phenylalanine ammonia lyase in catalyzing 9-anthracene acrylic acid (English name: 9-anthraceneacrylic acid, CAS number: 5335-33-1) to produce the product 3-(9-anthryl) alanine (English name: 3-(9-anthryl) alanine, CAS number: 100896-08-0) is very low. Therefore, there is currently a lack of enzymes that can efficiently catalyze 9-anthraceneacrylic acid to produce the product 3-(9-anthryl) alanine, as well as methods for screening enzyme mutants with higher catalytic activity from high-throughput enzyme libraries. Summary of the invention

[0006] In view of the above problems, the present invention provides a phenylalanine aminolyase mutant and its application. The present invention obtains a phenylalanine aminolyase mutant with high specificity and high catalytic activity by enzyme mutation and screening, and the mutant is applied in the field of non-natural amino acid synthesis to efficiently catalyze 9-anthracene acrylic acid to generate 3-(9-anthryl)alanine.

[0007] The present invention provides the use of a phenylalanine ammonia lyase mutant as a catalyst for catalyzing the generation of 3-(9-anthryl)alanine from 9-anthracene acrylic acid. The phenylalanine ammonia lyase mutant is a variant obtained by mutating one or more of the following amino acid substitution sites based on EU numbering, using the amino acid sequence of phenylalanine ammonia lyase E1-101-2-3 as a starting sequence: L134V, L138A, L256A, T114N, Q485S, Q485N, K393S, K393N, K336S, K105N, S102A, S102V, S109A, N392V, D101A, K105S, N384A, G115A, G111A, L134A, F137G, F137A, F137V, L138V, L20 6A, L206V, L256V, I460A, I460V, L138A, L134V, T114S, T114N, P385S, P385N, H396S, H396N.

[0008] Preferably, the phenylalanine ammonia lyase E1-101-2-3 has the amino acid sequence shown in SEQ ID NO:1.

[0009] Preferably, the phenylalanine ammonia lyase mutant is a variant obtained by mutating the amino acid substitution site F137G or L138V based on the EU numbering, based on the amino acid sequence of phenylalanine ammonia lyase E1-101-2-3.

[0010] Preferably, the phenylalanine ammonia lyase mutant has the amino acid sequence shown in SEQ ID NO: 82 or SEQ ID NO: 105.

[0011] Preferably, the catalytic activity of the phenylalanine ammonia lyase mutant having the amino acid sequence shown in SEQ ID NO:82 is higher than that of the phenylalanine ammonia lyase mutant having the amino acid sequence shown in SEQ ID NO:105.

[0012] Beneficial Effects

[0013] 1. The F137G and L138V mutants screened by the present invention can catalyze the substrate 9-anthracene acrylic acid to generate the product 3-(9-anthryl)alanine with a high conversion rate.

[0014] 2. The method for screening enzyme mutants of the present invention is efficient and sensitive. DETAILED DESCRIPTION

[0015] The present invention is further described by the following embodiments. It should be understood that the following embodiments are only used to illustrate the present invention, but not to limit the present invention.

[0016] The substrate 9-anthracene acrylic acid has three benzene rings, which makes the molecule highly hydrophobic, and the steric hindrance of the molecule is large, which makes it difficult to enter the active pocket of the enzyme, which poses a challenge to the construction of PAL mutant enzymes. Moreover, the yield of synthesizing 3-(9-anthryl)alanine by chemical means in the prior art is low and the cost is high. The present invention provides a phenylalanine aminolyase mutant that can efficiently catalyze the synthesis of non-natural amino acids and its application. The present invention efficiently screens out enzyme mutants that can catalyze the substrate 9-anthracene acrylic acid to generate the product 3-(9-anthryl)alanine from the phenylalanine aminolyase mutant library. Specifically, the mutants of phenylalanine ammonia lyase (enzyme 1-101-2-3) are used as the screening target, and the catalytic reaction of 9-anthracene acrylic acid to generate the product 3-(9-anthryl)alanine is carried out to screen active mutants.

[0017] The following examples are further listed to illustrate the present invention in detail. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention belong to the scope of protection of the present invention. The specific process parameters and the like in the following examples are also only examples within a suitable range, that is, those skilled in the art can make a selection within a suitable range through the description herein, and are not limited to the specific values ​​​​exemplified below.

[0018] Experimental Materials

[0019] Table 1

[0020]

[0021] Example 1: Construction of mutants

[0022] The synthesized E1-101-2-3 sequence was connected to the plasmid pET-28a (+) to obtain a recombinant plasmid containing E1-101-2-3. The recombinant plasmid was used as a template to construct the corresponding mutant. The conventional PCR site-directed mutagenesis technique was used to construct the mutant of phenylalanine ammonia lyase E1-101-2-3. The Quikchange site-directed mutagenesis kit was specifically used for mutant construction. The detailed operation was performed according to the instructions.

[0023] Amino acid sequence of phenylalanine ammonia lyase E1-101-2-3 (SEQ ID NO: 1):

[0024] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQ WLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVK NTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0025] Table 2

[0026]

[0027] Table 3

[0028]

[0029] Prepare the PCR system for site-directed mutagenesis as shown in Table 2 and mix well. Then perform the PCR reaction on the PCR instrument according to the PCR operation procedure shown in Table 3. The PCR product is treated with Dpn I enzyme at 37°C for 2 hours to remove the methylated plasmid template. Take 5 μL of the PCR product digested with Dpn I and transfer it into 100 μL of E.coli BL21 (DE3) super competent cells by chemical method, pick a single clone and extract the plasmid and sequence it to obtain the corresponding recombinant plasmid. The recombinant strain containing the correctly sequenced recombinant plasmid is stored in glycerol bacteria.

[0030] Table 4

[0031]

[0032]

[0033]

[0034]

[0035] Amino acid sequence of phenylalanine aminolyase proenzyme mutant

[0036] Amino acid sequence of the F137G single point mutant E1-101-2-3-F137G of phenylalanine ammonia lyase E1-101-2-3 (SEQ ID NO: 82):

[0037] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIR GLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0038] Amino acid sequence of the L138V single-point mutant E1-101-2-03-L138V of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:83):

[0039] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIRF VNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0040] Amino acid sequence of the L134V single point mutant E1-101-2-03-L134V of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:84):

[0041] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKE VIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0042] Amino acid sequence of the L138A single-point mutant E1-101-2-03-L138A of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:85):

[0043] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIRF ANAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0044] Amino acid sequence of the L256A single-point mutant E-1-101-2-3-L256A of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:86):

[0045] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEG AALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0046] Amino acid sequence of the single-point mutant E1-101-2-3-T114N of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:87):

[0047] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVT NGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0048] Amino acid sequence of the Q485S single-point mutant E1-101-2-3-Q485S of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:88):

[0049] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAE S HNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0050] Amino acid sequence of the single-point mutant E1-101-2-3-Q485N of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:89):

[0051] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAE N HNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0052] Amino acid sequence of the single-point mutant E1-101-2-3-K393S of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:90):

[0053] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRN S AIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0054] Amino acid sequence of the single-point mutant E1-101-2-3-K393N of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:91):

[0055] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRN N AIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0056] Amino acid sequence of the K336S single point mutant E1-101-2-3-K336S of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:92):

[0057] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQ S LHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0058] Amino acid sequence of the single-point mutant E1-101-2-3-K105N of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:93):

[0059] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMN NGTDSYGVTTGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0060] Amino acid sequence of the S102A single point mutant E1-101-2-3-S102A of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:94):

[0061] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMD AMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0062] Amino acid sequence of the single-point mutant E1-101-2-3-S102V of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:95):

[0063] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMD VMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0064] Amino acid sequence of the S109A single point mutant E1-101-2-3-S109A of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:96):

[0065] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTD AYGVTTGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0066] Amino acid sequence of the N392V single point mutant E1-101-2-3-N392V of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:97):

[0067] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSV V DNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0068] Amino acid sequence of the D101A single-point mutant E1-101-2-3-D101A of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:98):

[0069] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVM ASMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0070] Amino acid sequence of the single-point mutant E1-101-2-3-K105S of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:99):

[0071] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMN SGTDSYGVTTGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0072] Amino acid sequence of the N384A single point mutant E1-101-2-3-N384A of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:100):

[0073] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVND A PLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0074] Amino acid sequence of the G115A single-point mutant E1-101-2-3-G115A of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:101):

[0075] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTT AFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0076] Amino acid sequence of the G111A single point mutant E1-101-2-3-G111A of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:102):

[0077] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSY AVTTGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0078] Amino acid sequence of the L134A single point mutant E1-101-2-3-L134A of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:103):

[0079] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKE AIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0080] Amino acid sequence of the F137A single-point mutant E1-101-2-3-F137A of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:104):

[0081] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIR ALNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0082] Amino acid sequence of the L138V single-point mutant E1-101-2-3-L138V of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:105):

[0083] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIRF VNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0084] Amino acid sequence of the L206A single-point mutant E1-101-2-3-L206A of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:106):

[0085] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGD AVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0086] Amino acid sequence of the L206V single-point mutant E1-101-2-3-L206V of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:107):

[0087] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGD VVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0088] Amino acid sequence of the L256V single point mutant E1-101-2-3-L256V of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:108):

[0089] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEG VALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0090] Amino acid sequence of the I460A single point mutant E1-101-2-3-I460A of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:109):

[0091] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAE A AMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0092] Amino acid sequence of the I460V single-point mutant E1-101-2-3-I460V of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:110):

[0093] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAE V AMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0094] Amino acid sequence of the F137A / L138V combinatorial mutant E1-101-2-3-F137A / L138V of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:111):

[0095] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIRAV NAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0096] Amino acid sequence of the F137A / L138A combined mutant E1-101-2-3-F137A / L138A of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:112):

[0097] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIR AANAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0098] Amino acid sequence of the F137V / L138A combinatorial mutant E1-101-2-3-F137V / L138A of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:113):

[0099] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIR VANAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0100] F137V / L138V combinatorial mutant E1-101-2-3-F137V / L138V of phenylalanine ammonia-lyase E1-101-2-3

[0101] Amino acid sequence (SEQ ID NO:114):

[0102] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIR VVNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0103] L134V / F137A combinatorial mutant E1-101-2-3-L134V / F137A of phenylalanine ammonia-lyase E1-101-2-3

[0104] Amino acid sequence (SEQ ID NO:115):

[0105] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKE V IR ALNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0106] Amino acid sequence of the single-point mutant E1-101-2-3-T114S of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:116):

[0107] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVT SGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0108] Amino acid sequence of the T114N single-point mutant E1-101-2-3-T114N of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:117):

[0109] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVT NGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0110] Amino acid sequence of the P385S single-point mutant E1-101-2-3-P385S of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:118):

[0111] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDN S LIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0112] Amino acid sequence of the P385N single-point mutant E1-101-2-3-P385N of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:119):

[0113] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDN N LIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0114] Amino acid sequence of the H396S single-point mutant E1-101-2-3-H396S of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:120):

[0115] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAI S GGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0116] Amino acid sequence of the H396N single-point mutant E1-101-2-3-H396N of phenylalanine ammonia-lyase E1-101-2-3 (SEQ ID NO:121):

[0117] MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLGGETLTISQVAAISARDGSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIRFLNAGIFGNGSDNTLPHSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSAIFAEVMQGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAI N GGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFLVGLCQAIDLRHLEENLKSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQKLRQTLVEHALKNGDNERNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKELGTEYLTGEKVTSPGEEFEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

[0118] The F137G single-point mutant E1-101-2-3-F137G of phenylalanine ammonia-lyase E1-101-2-3 (nucleotide sequence of E1-101-2-3 (SEQ ID NO:122)):

[0119]

[0120] Example 2: Screening of mutants

[0121] The mutants of phenylalanine ammonia lyase (enzyme E1-101-2-3) were selected as the screening target, and the mutants that can catalyze the reaction to synthesize unnatural amino acids were screened out. Specifically, the mutants of phenylalanine ammonia lyase (enzyme E1-101-2-3) were selected as the screening target, and the active mutants that can efficiently catalyze 9-anthracene acrylic acid to produce the product 3-(9-anthryl) alanine were screened out. In this reaction system, the amino group needs to be introduced by enzyme catalysis, and ammonia water is used as the amino group donor for the catalytic reaction.

[0122]

[0123] 2.1. Cell culture and enzyme expression

[0124] Take 100 μL of the glycerol bacteria prepared above and add it to 3 mL of LB medium containing antibiotics (50 ng / μL final concentration of kanamycin). Place it in a shaker at 37°C and 220 rpm until the OD reaches 600 The value is 0.6.

[0125] Take 100 μL of the above bacterial solution and add it to 100 mL of LB medium containing antibiotics (50 ng / μL final concentration of kanamycin). Place it in a shaker at 37°C and 220 rpm until the OD reaches 600 The value reached about 0.3. Then the shaker was set to 16℃ and 180 rpm to continue the culture. 600 When it reaches about 0.6, add IPTG with a final concentration of 0.5 mM, place in a shaker at 16°C, 220 rpm, and continue overnight culture for about 20 hours.

[0126] 2.2 Cell lysis and protein concentration determination

[0127] (1) Preparation of cell lysis buffer A: 100 mM Tris-HCl (pH 7.4), 100 mM NaCl, 10% glycerol.

[0128] (2) Centrifuge 100 mL of overnight bacterial solution at 6000 rpm and 4°C for 10 minutes, discard the supernatant and keep the precipitate. Resuspend the bacterial solution with 30 mL of cell lysis buffer A, then centrifuge the resuspended bacterial solution at 6000 rpm and 4°C for 10 minutes, discard the supernatant, and keep the precipitate for direct crushing or storage in a -80°C refrigerator.

[0129] (3) Resuspend the bacterial solution in 5 mL of cell lysis buffer A (thaw the solution on ice before resuspending). Place the resuspended bacterial solution in an ice water bath and sonicate at 300 W for 2 seconds, stop for 5 seconds, and then sonicate for 15 minutes.

[0130] (4) After the cells are ultrasonically disrupted, centrifuge at 12,000 rpm and 4°C for 45 minutes, and then transfer the supernatant to a new centrifuge tube pre-cooled on ice to obtain the cell lysate. The cell lysate is divided into small tubes and stored in a -80°C refrigerator for later use.

[0131] (5) Use the BCA protein concentration assay kit to determine the protein concentration of the cell lysate.

[0132] 2.3 Catalytic reaction experiment

[0133] The volume of the reaction system is 1 mL, and it is carried out in a 2 mL centrifuge tube. Prepare buffer B in advance: 100 mMTris (pH 8.8).

[0134] Table 5 Reaction system

[0135]

[0136] The substrate, ammonia and buffer B were premixed, and then the enzyme mutant was added to start the reaction. The reaction was incubated at 30°C and 200 rpm for about 20 hours.

[0137] 2.4 Enzyme removal and HPLC detection of products and substrates

[0138] After the incubation, boil for about 10 minutes to inactivate the enzyme, centrifuge at 12,000 rpm for 8 minutes (for centrifuge tube catalytic system), and take the supernatant.

[0139] The supernatant was filtered through a 0.22 μm filter membrane into a liquid phase vial, and then the substrate and product were detected by HPLC. The liquid phase detection conditions were as follows:

[0140] Column: XBRIDGE C18 50*4.6mm 5μm;

[0141] Mobile phase: solution A was 10 mM NH4HCO3·H2O, solution B was acetonitrile, flow rate was 2 mL / min;

[0142] Flow conditions: 10% solution B for 0.4 minutes, then the concentration of solution B was increased to 95% in 1.6 minutes, 95% solution B for 0.9 minutes, and then the concentration of solution B was reduced to 10% in 0.01 minutes;

[0143] Column temperature: maintained at 40°C;

[0144] Injection volume: 5 μL.

[0145] LC-MS analysis was performed on the substrate 9-anthracene acrylic acid, the product 3-(9-anthryl)alanine, and the substrate + product. The peak area of ​​the product in the LC-MS analysis spectrum was used to determine whether the mutant had catalytic activity. If the product peak area was large, the catalytic activity of the enzyme mutant was large.

[0146] Table 6

[0147]

[0148] Table 6 shows the catalytic activity results of the enzyme mutants. It can be seen that the two mutants F137G and L138V obtained by screening can efficiently catalyze the substrate 9-anthracene acrylic acid to produce the product 3-(9-anthryl)alanine.

[0149] LC-MS analysis was performed on the enzyme mutant F137G catalyzing 9-anthracene acrylic acid to produce 3-(9-anthryl)alanine. In positive ion mode, the sample had a signal peak of M / Z=266 [M+1]+, which was consistent with the information obtained from the test at 11.228 minutes, so it was inferred that the molecule with the peak at this retention time was the target product.

[0150]

[0151] LC-MS analysis of the enzyme mutant L138V catalyzing 9-anthracene acrylic acid to produce 3-(9-anthryl)alanine.

[0152] After calculation, the conversion rates (percentage of reactant conversion) of the two mutants F137G and L138V were Conv.=17% and Conv.=1.6%, respectively, which is consistent with the catalytic activity results in Table 3.

Claims

1. A use of a phenylalanine ammonia lyase mutant as a catalyst for catalyzing the production of 3-(9-anthryl)alanine from 9-anthracene acrylic acid, characterized in that: The phenylalanine ammonia lyase mutant is a variant obtained by mutating the amino acid substitution site F137G or L138V based on the EU numbering, with the amino acid sequence of phenylalanine ammonia lyase E1-101-2-3 as the starting sequence; wherein the phenylalanine ammonia lyase E1-101-2-3 is the amino acid sequence shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: The phenylalanine ammonia lyase mutant has an amino acid sequence shown in SEQ ID NO:

82.

3. The use according to claim 1, characterized in that: The phenylalanine ammonia lyase mutant has an amino acid sequence shown in SEQ ID NO:

105.

4. The use according to claim 1, characterized in that: The catalytic activity of the phenylalanine ammonia lyase mutant having the amino acid sequence shown in SEQ ID NO:82 is higher than that of the phenylalanine ammonia lyase mutant having the amino acid sequence shown in SEQ ID NO:105.

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