Pal variants, pharmaceutical compositions comprising the pal variants and methods for preparing the pal variants

Abstract

The invention provides a variant of phenylalanine ammonialyase (PAL), the phenylalanine ammonialyase (PAL) is derived from variant anabaena and contains an amino acid sequence as shown in SEQ ID NO: 1, the variant contains amino acid substitution C503S / C565L, so that the variant contains the amino acid sequence as shown in SEQ ID NO: 2; or amino acid substitutes C503S / C565P, so that the variant comprises an amino acid sequence as shown in SEQ ID NO: 3; or amino acid substitutes C503L / C565P, so that the variant comprises an amino acid sequence as shown in SEQ ID NO: 4. The invention also provides a conjugate and a pharmaceutical composition comprising the variant, a polynucleotide for encoding the variant, a bacterium comprising the variant and a method for preparing the variant.

Description

PAL variants, pharmaceutical compositions containing the same, and methods for preparing the same CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority and benefits of Chinese Patent Application No. 202210839871.7 filed with the State Intellectual Property Office of China on July 18, 2022, the entire contents of which are incorporated herein by reference. Technical Field The present invention provides a variant of phenylalanine ammonia lyase (PAL) derived from Anabaena variabilis, a conjugate and a pharmaceutical composition comprising the variant, a polynucleotide encoding the variant, a bacterium comprising the variant and a method for preparing the variant. Background Art Phenylalanine ammonia lyase (PAL) is a member of the aromatic amino acid lyase family (EC 4.3.1.23-1.25 and 4.3.1.3) together with histidine ammonia lyase (HAL) and tyrosine ammonia lyase (TAL). More specifically, enzymes with PAL activity (EC 4.3.1.23-1.25 and previously classified as EC4.3.1.5) catalyze the non-oxidative deamination of L-phenylalanine to trans-cinnamic acid. PAL is a non-mammalian enzyme that is widely distributed in plants, fungi, and a limited number of bacteria. PAL enzymes can be used as therapeutic proteins for the treatment of the metabolic disorder phenylketonuria (PKU). PKU is an autosomal metabolic genetic disorder in which the liver enzyme phenylalanine hydroxylase (PAH) or one or more enzymes involved in the synthesis or recycling of the cofactor tetrahydrobiopterin lack function due to mutations in the corresponding genes. This lack of function leads to higher levels of phenylalanine in the bloodstream. Higher levels of phenylalanine are converted to phenylpyruvate (phenyl ketone) and other derivatives. If PKU is not treated promptly, high levels of phenylalanine and some of its breakdown products in the human body can cause significant medical problems, including intellectual disability, microcephaly, and epileptic seizures. Many studies have focused on the use of PAL for the treatment of PKU through enzyme replacement (Ambrus et al., Science 201:837-839).

[1978] ; Bourget et al., Appl Biochem. Biotechnol, 10:57-59

[1984] ; and Sarkissian et al., Proc. Natl. Acad. Sci. USA 96:2339-2344

[1999] ). One method for removing phenylalanine from serum is to use injectable recombinant PAL and PAL variants modified by pegylation (PEG-PAL). The pegylation method has been shown to improve the half-life of the enzyme and reduce the antigenic response of the subject (see WO 2008 / 153776, WO 2011 / 097335 and US7531341). PAL variants that are effective in PEG-PAL compositions have been described as wild-type Nostoc punctiforme (NpPAL); variants of Anabaena variabilis (PAL) and Rhodosporidium toruloides (RtPAL). In particular, variants of wild-type PAL have been described in which the cysteine ​​residues at positions 64, 318, 503 and 565 are replaced by serine (see US7790433; US7560263 and US7537923). Although PAL has the potential for various therapeutic uses, factors such as reduced specific activity and proteolytic instability have limited the use of PAL. Similar to other therapeutic proteins, the use of PAL as an enzyme therapy is accompanied by several disadvantages, such as immunogenicity and proteolytic sensitivity (see Vellard, Curr. Opin. Biotechnol. 14: 1-7 (2003)). In addition, a delicate balance between substrate affinity and enzyme activity is required to achieve and maintain plasma phenylalanine levels within a normal but somewhat narrow range in diseases characterized by hyperphenylalaninemia. To date, due to the lack of structural and biochemical knowledge about this protein, no concerted efforts have been made to improve these parameters. Therefore, there remains a need for PAL molecules with optimal kinetic characteristics (including potent catalytic activity, longer biological half-life, greater biochemical stability and / or reduced immunogenicity) for therapeutic uses, such as the treatment of hyperphenylalaninemia (HPA, such as PKU) and other diseases (such as cancer). Summary of the invention In order to solve the above problems, the object of the present invention is to provide a phenylalanine ammonia lyase (PAL) variant according to the present invention or obtained by the method according to the present invention, which is a variant of Anabaena phenylalanine ammonia lyase (UniProtKB / Swiss Prot: Q3M5Z3.1) having an amino acid sequence shown in SEQ ID NO: 1, or the variant has an amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. The PAL variant according to the present invention may include one or more amino acid substitutions or amino acid substitution combinations, for example, a combination of one or more of the following: C503S / C565L, C503S / C565P, C503L / C565P mutations, and T102R, T102H, I165L, I165V, G166A, G166S, G218A, G218S , G218N, M222L, M222I, N437S, S438N, S438A, I439V, I439C, I439A, I439K, A440T, A440G, A440V, A440T, R441N, R442Q, T445V, T445S, A447S, E448K, N453G, Q457L and G458K. In particular, the PAL variant enzyme according to the present invention has phenylalanine ammonia lyase activity. The PAL variant enzyme according to the invention or obtained by the method according to the invention can be used to treat diseases or disorders characterized by elevated blood concentrations of phenylalanine. In particular, the PAL variant enzyme according to the invention can be used to treat phenylketonuria (PKU). According to one embodiment of the present invention, the PAL variant enzyme according to the present invention has enhanced catalytic activity from phenylalanine to trans-cinnamic acid, enhanced thermal stability and low aggregation. The definitions set forth below and throughout this application are used herein to describe the subject matter in accordance with the present invention. Definitions and terminology The following definitions and terms may be used to further describe the disclosed subject matter. The definitions and terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting. In the context of the present invention, “approximately”, “about”, “substantially ... “Substantially,” “substantially,” and “significantly” will be understood by one of ordinary skill in the art and will depend to some extent on the context in which they are used. If the use of a term is unclear to one of ordinary skill in the art in the context in which the term is used, “approximately” and “about” will mean plus or minus 10% of the particular term, while “substantially” and “significantly” will mean more than plus or minus 10% of the particular term. In the context of the present invention, the terms "comprising" and "including" should be interpreted as open, allowing the inclusion of additional parts on the basis of those listed. The terms "consisting of" and "composed of" should be interpreted as closed, not allowing the inclusion of other parts except those listed in the claims. The term "consisting essentially of..." should be interpreted as partially closed, that is, only additional components that will not fundamentally change the properties of the claimed target substance are allowed to be included. The modal verb "may" or "might" refers to the preferred use or selection of one or more options or choices among the several embodiments or features described. If no options or choices are disclosed regarding the specific embodiments or features contained therein, the modal verb "may" refers to an affirmative action regarding how to perform or use the embodiments or features described. In the following context, the modal verb "may" has the same meaning and connotation as the auxiliary verb "may". In the context of the present invention, the following amino acid abbreviations are used: Alanine: Ala(A) Arginine: Arg(R) Asparagine: Asn(N) Aspartic acid: Asp(D) Cysteinyl: Cys(C) Glutamine: Gln(Q) Glutamic acid: Glu(E) Glycine: Gly(G) Histidine: His(H) Isoleucine: Ile(I) Leucine: Leu(L) Lysine: Lys(K) Methionine: Met(M) Phenylalanine: Phe(F) Proline: Pro(P) Serine: Ser(S) Threonine: Thr(T) Tryptophan: Trp(W) Tyrosine: Tyr(Y) Valyl: Val(V) In the context of the present invention, the terms "peptide", "polypeptide" and "protein" refer to molecules comprising a polymer of chains of amino acid residues linked by amide bonds. The term "amino acid residue" includes, but is not limited to, amino acid residues contained in the group consisting of alanine (Ala or A), cysteine ​​(Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (lie or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W) and tyrosine (Tyr or Y). The term "amino acid residue" may also include non-standard or non-natural Amino Acids. The term "amino acid residue" may include α, β, γ and δ amino acids. In the context of the present invention, "protein", "polypeptide" and "peptide" are used interchangeably herein to refer to a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modifications (eg, glycosylation or phosphorylation). As used in the context of the present invention, an "analog" or "derivative" is a compound, such as a peptide, having a sequence similarity greater than about 70% but less than 100% with a given compound, such as a peptide. These analogs or derivatives may contain non-naturally occurring amino acid residues as well as naturally occurring amino acid residues. These analogs or derivatives may also contain one or more D-amino acid residues and may contain non-peptide linkages between two or more amino acid residues. In the context of the present invention, "polynucleotide" refers to a polymer composed of nucleotide units. Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and nucleic acid analogs. Nucleic acid analogs include non-natural bases, nucleotides that participate in bonds with other nucleotides other than naturally occurring phosphodiester bonds, or bases connected by bonds other than phosphodiester bonds. Therefore, nucleotide analogs include, for example but not limited to, phosphorothioates, phosphorodithioates, phosphotriesters, phosphoramidates, borane phosphates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNA), etc. For example, these polynucleotides can be synthesized using an automatic DNA synthesizer. The term "nucleic acid" generally refers to large polynucleotides. The term "oligonucleotide" generally refers to short polynucleotides, usually not more than about 50 nucleotides. It should be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C), wherein "U" replaces "T". According to one embodiment of the present invention, there is provided a vector, such as an expression vector, containing a nucleic acid encoding one or more polypeptides and / or proteins according to the present invention. In the context of the present invention, the term "coding" refers to the inherent properties of other polymers and macromolecules in the synthesis of biological processes, such as specific sequences of nucleotides in polynucleotides such as genes, cDNAs or mRNAs, which are used as templates, and these polymers and macromolecules have a determined nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a determined amino acid sequence and the biological properties produced therefrom. Therefore, if in a cell or other biological system, the transcription and translation of the mRNA produced by a gene produces a protein, then the gene encodes the protein. The coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence table) and the non-coding strand used as the transcription template of a gene or cDNA can be referred to as encoding the protein or other product of the gene or cDNA. Unless otherwise stated, "nucleotide sequences encoding amino acid sequences" include all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. The nucleotide sequences encoding proteins and RNA can only contain introns. In the context of the present invention, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked, and an "expression vector" refers to a vector comprising a recombinant polynucleotide that is operably linked to a nucleotide sequence to be expressed. The expression vector comprises a cis-acting element sufficient for expression; other expression elements may be provided by the host cell or an in vitro expression system. The expression vector includes all expression vectors known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses into which the recombinant polynucleotide has been integrated. In the context of the present invention, the term "recombinant polynucleotide" refers to a polynucleotide having sequences that are not linked together in nature. The amplified or assembled recombinant polynucleotide can be contained in a suitable vector, and the vector can be used to transform a suitable host cell. A host cell containing a recombinant polynucleotide is called a "recombinant host cell". The gene is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide". Recombinant polynucleotides can also play non-coding functions (e.g., promoters, replication origins, ribosome binding sites, etc.). In one embodiment according to the invention, the expression vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. In the context of the present invention, "insertion" refers to adding one or more nucleotides to a native polynucleotide sequence. Engineered strains disclosed herein may include inserting one or more genes. In certain embodiments, engineered strains disclosed herein include inserting sequences encoding exogenous phenylalanine ammonia lyase (PAL). In certain embodiments, engineered strains disclosed herein include insertions (i.e., genomic insertions) in endogenous genes that result in non-functional gene products. In the context of the present invention, the term "replacement" refers to replacing the nucleotides of a native polynucleotide sequence with non-natural nucleotides of the polynucleotide sequence. Engineered strains disclosed herein may include replacements in one or more genes. In certain embodiments, replacements result in non-functional gene products, for example, wherein replacements introduce premature stop codons (for example, TAA, TAG or TGA) in the coding sequence of a gene product. In some embodiments according to the present invention, engineered strains according to the present invention may include two or more replacements, wherein replacements introduce multiple premature stop codons (for example TAATAA, TAGTAG or TGA). In the context of the present invention, the term "primer" refers to a polynucleotide that can specifically hybridize with a specified polynucleotide template and provide a starting point for synthesis of a complementary polynucleotide. When a polynucleotide primer is placed under conditions of induction synthesis, that is, in the presence of nucleotides, complementary polynucleotide templates and polymerization reagents such as DNA polymerase, this synthesis occurs. Primers are generally single-stranded, but can also be double-stranded. Primers are generally deoxyribonucleic acids, but many types of synthetic and naturally occurring primers can also be used for many applications. Primers are complementary to the templates designed to hybridize therewith, to be used as the site for starting synthesis, but do not need to reflect the exact sequence of the template. In this case, the specific hybridization of primers and templates depends on the stringency of hybridization conditions. Primers can be labeled with, for example, chromogenic, radioactive or fluorescent moieties, and are used as detectable moieties. In the context of the present invention, the term "wild type" refers to the natural genetic form of an organism. The wild type is distinct from a mutant form (an organism with a genetic mutation). In the context of the present invention, "variants according to the present invention", "PAL variants", "variants of phenylalanine ammonia lyase", "variant PAL", "PAL variant enzyme" and "PAL enzyme variant" are used interchangeably. In the context of the present invention, the term "effective amount" refers to a dosage sufficient to produce a desired result for an individual's health condition, pathology, and disease or for diagnostic purposes. The desired result may include a subjective or objective improvement in the recipient of the dose. A "therapeutically effective amount" refers to an amount of a drug that is effective to produce a desired beneficial effect on health. In any individual case, an appropriate "effective" amount can be determined by those skilled in the art using routine experimentation. In the context of the present invention, the term "treatment" refers to prophylactic treatment or therapeutic treatment or diagnostic treatment. In the context of the present invention, the term "pharmaceutical composition" refers to a composition suitable for use as a drug in an animal subject, including humans and mammals. The pharmaceutical composition comprises a pharmacologically effective amount of a PAL polypeptide and also comprises a pharmaceutically acceptable carrier. The pharmaceutical composition includes an active ingredient and an inert component constituting a carrier, as well as any product produced directly or indirectly by the combination, complex or aggregation of any two or more components, or the dissociation of one or more components, or other types of reactions or interactions of one or more components. Therefore, the pharmaceutical composition of the present invention includes any composition prepared by mixing the compound or conjugate of the present invention with a pharmaceutically acceptable carrier. In the context of the present invention, the term "pharmaceutically acceptable carrier" refers to any of standard pharmaceutical carriers, buffers and excipients (e.g., phosphate buffered saline, 5% dextrose in water) and emulsions (e.g., water-in-oil or oil-in-water emulsions) and various types of wetting agents and / or adjuvants. In a first aspect of the present invention, a PAL variant, PAL variant enzyme, PAL mutant or analogue according to the present invention is provided. According to one embodiment of the present invention, the PAL is derived from Anabaena variabilis and has the amino acid sequence shown in SEQ: ID NO: 1, and the PAL variant according to the present invention has an amino acid substitution selected from any one or a combination of two or more of the following: C503S / C565L, C503S / C565P, C503L / C565P, T102R, T102H, I165L, I165V, G166A, G166S, G218A, G218S, G 457S, A457L, A457K, N453G, Q457L and G458K. Preferably, the PAL variant enzyme according to the present invention has phenylalanine ammonia lyase activity (EC 4.3.1.24). Preferably, the PAL variant enzyme according to the present invention has an enhanced phenylalanine ammonia lyase activity (EC 4.3.1.24) relative to the wild-type PAL enzyme. With the help of the PAL variant according to the present invention, an enhancement of the catalytic activity from phenylalanine to trans-cinnamic acid, an enhancement of the thermal stability and a low aggregation are achieved. According to one embodiment of the present invention, the PAL variant according to the present invention comprises the amino acid substitutions C503L / C565P / 222L, or the amino acid substitutions C503L / C565P / G218A. According to one embodiment of the present invention, in the reaction of converting phenylalanine into trans-cinnamic acid, the activity of the variant is higher than that of the phenylalanine ammonia lyase comprising the amino acid sequence shown in SEQ ID NO: 1. According to one embodiment of the present invention, the variant exhibits the same stability or higher stability at a temperature in the range of 37°C to 70°C compared to the phenylalanine ammonia lyase comprising the amino acid sequence shown in SEQ ID NO:1. In other words, at a temperature in the range of 37°C to 70°C, the PAL variant according to the present invention has the same or higher, and preferably higher, stability than the wild-type PAL. According to one embodiment of the present invention, the variant has low aggregation compared to the phenylalanine ammonia lyase comprising the amino acid sequence shown in SEQ ID NO:1. According to one embodiment of the present invention, the PAL variant or derivative according to the present invention may have an amino acid sequence that includes conservative amino acid substitutions or non-conservative amino acid substitutions relative to the reference amino acid sequence SEQ: ID NO: 1. For example, the PAL variant or derivative peptide, polypeptide or protein according to the present invention may include conservative amino acid substitutions and / or non-conservative amino acid substitutions relative to the reference peptide, polypeptide or protein. "Conservative amino acid substitutions" refer to substitutions that are expected to have the least interference with the properties of the reference peptide, polypeptide or protein. For example, each of the following six groups includes amino acids that are conservative substitutions for each other: (1) alanine (A), serine (S), threonine (T); (2) aspartic acid (D), glutamic acid (E); (3) asparagine (N), glutamic acid (Q); (4) arginine (R), lysine (K); (5) isoleucine (I), leucine (L), methionine (M), valine (V); and (6) phenylalanine (F), tyrosine (Y), tryptophan (W). "Non-conservative amino acid substitutions" refer to substitutions that are expected to have the greatest interference with the properties of the reference peptide, polypeptide or protein. In other words, conservative amino acid substitutions substantially preserve the structure and function of the reference peptide, polypeptide or protein, whereas non-conservative amino acid substitutions do not preserve the structure and function of the reference peptide, polypeptide or protein. According to one embodiment of the present invention, the PAL variant according to the present invention can be expressed in plants, fungi and bacteria. According to a preferred embodiment of the present invention, the PAL variant according to the present invention can be expressed in Nostoc punctata, Anabaena variabilis, Rhodosporidium toruloides, Streptomyces maritima, Anastrozole nidulans, Photorhabditis luminescens, Streptomyces verticillatus, and Escherichia coli. According to one embodiment of the present invention, the PAL variant according to the present invention can be expressed in a probiotic or engineered bacterium that is capable of polynucleotide recombination and is not a pathogenic bacterium and does not produce toxins. The PAL variant according to the present invention can be produced by introducing a recombinant polynucleotide encoding the PAL variant according to the present invention into the probiotic or engineered bacterium and expressing it. Examples of the probiotic or engineered bacterium include Escherichia coli and G. + Bacteria, such as Bacillus subtilis. In a second aspect of the present invention, the present invention provides a conjugate comprising a PAL variant according to the present invention, wherein the conjugate comprises a PAL variant according to the present invention and a suitable polymer, and in particular consists of a PAL variant according to the present invention and a suitable water-soluble polymer. Here, the suitable water-soluble polymer includes polyethylene glycol, polyzwitterionic polymers, and polyoxazoline. According to a preferred embodiment of the present invention, the water-soluble polymer is polyethylene glycol. In a third aspect of the present invention, the present invention provides a pharmaceutical composition comprising: (i) a PAL variant enzyme according to the present invention; and (ii) a suitable pharmaceutically acceptable carrier. According to one embodiment of the present invention, the pharmaceutical composition according to the present invention comprises: (i) a conjugate of a PAL variant enzyme according to the present invention; and (ii) a suitable pharmaceutically acceptable carrier. It is contemplated that the conjugate of the PAL variant enzyme comprises a PAL variant enzyme chemically coupled to a polyethylene glycol (PEG) polymer. According to another embodiment of the present invention, the PAL variant in the pharmaceutical composition according to the present invention has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:4. According to one embodiment of the present invention, a pharmaceutical composition according to the present invention is prepared and administered to a subject in need thereof. Taking into account factors such as age, sex, body weight and condition of a particular patient and route of administration, the pharmaceutical composition according to the present invention can be prepared and / or administered according to dosage and technology well known to those skilled in the art of medicine. According to one embodiment of the present invention, the pharmaceutical composition according to the present invention comprises a pharmaceutically acceptable carrier, a diluent, an excipient and a surfactant. In addition, the pharmaceutical composition may also include a preservative (e.g., an antimicrobial or an antimicrobial, such as benzalkonium chloride). According to a preferred embodiment of the present invention, the pharmaceutical composition may also include a buffer (e.g., in order to maintain the pH of the composition between 6.5 and 7.5). According to one embodiment of the present invention, the pharmaceutical composition according to the present invention can be used to treat phenylketonuria. In the fourth aspect of the present invention, the present invention provides a polynucleotide for encoding a PAL variant according to the present invention, wherein the polynucleotide is DNA or RNA, and in particular, the polynucleotide is cDNA or mRNA or tRNA. These DNA and RNA have specific nucleotide sequences, so that the PAL variant enzyme according to the present invention can be synthesized using them as templates. According to one embodiment of the present invention, the polynucleotide according to the present invention is codon optimized so that the polynucleotide can be used to express the PAL variant according to the present invention in Escherichia coli. According to a preferred embodiment of the present invention, the polynucleotide according to the present invention is codon optimized so that the polynucleotide can be used to express the PAL variant according to the present invention in animal cells, especially human cells. According to a preferred embodiment of the present invention, the polynucleotide according to the present invention is codon-optimized so that the PAL variant enzyme according to the present invention can be expressed in E. coli. According to one embodiment of the present invention, the polynucleotide according to the present invention is codon-optimized so that the PAL variant enzyme according to the present invention can be expressed in human cells. The disclosed polynucleotides can be present in vectors known in the art (e.g., plasmid vectors). According to one embodiment of the present invention, when the coding gene of the PAL enzyme variant is inserted into the vector pET28a, the base "GC" is introduced after the start codon of the restriction site NcoI, that is, a glycine is introduced after the first methionine to prevent frameshift during translation. The PAL variants, for example, variants of the mutant Anabaena phenylalanine ammonia lyase PAL (SEQ ID NO: 1), for example, include Other PAL variants according to the present invention are PAL variants of the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4. In a fifth aspect of the present invention, an expression vector according to the present invention is provided, the expression vector comprises a polynucleotide according to the present invention, wherein the expression vector can be expressed in living cells. According to one embodiment of the present invention, the expression vector according to the present invention includes all expression vectors known in the art, such as cosmids, plasmids (eg, naked or contained in liposomes) and viruses incorporating recombinant polynucleotides. According to a preferred embodiment of the present invention, the expression vector according to the present invention is a viral vector, such as adenovirus, adeno-associated virus and lentivirus. According to another preferred embodiment of the present invention, the expression vector according to the present invention is a plasmid, which comprises a promoter, and the promoter is operably linked to the polynucleotide according to the present invention. According to another preferred embodiment of the present invention, the plasmid is transfected into cells via liposomes or cationic polymers. In a sixth aspect of the present invention, the present invention provides a bacterium according to the present invention, wherein the bacterium has a polynucleotide according to the present invention integrated into its genome and / or is capable of expressing a PAL variant according to the present invention. Here, the present invention also provides a modified cell according to the present invention, the modified cell comprising a PAL variant according to the present invention and / or a polynucleotide according to the present invention and / or an expression vector according to the present invention, wherein optionally, the modified cell is an animal cell, especially a human cell. In a host cell (e.g., a bacterium according to the invention or a modified cell according to the invention) comprising at least one polynucleotide encoding at least one PAL variant according to the invention, the polynucleotide is operably linked to one or more control sequences for expressing the PAL variant in the host cell. Suitable host cells for expressing the PAL variant according to the invention are, for example, bacteria according to the invention, such as Escherichia coli, Vibrio fluvialis, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris); insect cells, such as Drosophila S2 and Spodoptera Sf9 cells; animal cells, such as CHO, COS, BHK, 293 and Bowes melanoma cells; and plant cells. Exemplary host cells also include various Escherichia coli strains (e.g., W3110 (ΔfhuA) and BL21 (DE3)). In its seventh aspect, the present invention provides a method for preparing a PAL variant according to the present invention, the method comprising culturing the bacterium according to the present invention or the modified cell according to the present invention in a culture medium to produce the PAL variant according to the present invention and isolating the PAL variant according to the present invention from the modified cell and / or the culture medium. The PAL variant enzymes obtained by the process according to the invention can be used to treat diseases or disorders characterized by elevated blood concentrations of phenylalanine, such as phenylketonuria (PKU). According to one embodiment of the present invention, the PAL variant enzyme obtained by the method according to the present invention is compared to the PAL enzyme derived from Anabaena vulgaris (SEQ ID NO: 1) has one or more amino acid substitutions selected from the group consisting of C503S / C565L, C503S / C565P, C503L / C565P mutations and T102R, T102H, I165L, I165V, G166A, G166S, G218A, G218S, G218N, M222L, M222I, N437S, S438N, S438A, I439V, I439C, I439A, I439K, A440T, A440G, A440V, A440T, R441N, R442Q, T445V, T445S, A447S, E448K, N453G, Q457L and G458K. Specifically, according to one embodiment of the present invention, the method according to the present invention specifically comprises one or more of the following steps: (i) constructing a PAL original enzyme expression system including a plasmid, an expression cell or the like; (ii) extracting the pET28a-PAL plasmid from the PAL host cell; (iii) using the plasmid obtained in step (ii) as a template, constructing a mutant library using site-directed mutagenesis technology; (iv) cultivating PAL variants using a high-throughput screening method, and screening for superior mutations by detecting the enzyme activity of the variants. Components in the context of the present invention may be crude and / or at least partially separated and / or purified. In the context of the present invention, the term "separated or purified" may refer to components that are removed from their natural environment and / or medium and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components and / or the medium with which they are naturally associated. In the method for preparing the PAL variant according to the present invention, the PAL enzyme variants suitable for the method include, but are not limited to, variants of the PAL enzyme derived from Anabaena variabilis. Suitable transformed cells may include, but are not limited to, transformed E. coli, wherein the PAL gene sequence is preferably codon-optimized to express the PAL enzyme variant according to the present invention in E. coli. According to one embodiment of the present invention, the culture medium may be further supplemented and / or modified to promote the growth of transformed cells. According to one embodiment of the present invention, glucose is added to the culture medium, and the concentration may be 0.1-0.3% (v / v). According to one embodiment of the present invention, the transformed cells can be passaged one or more times. For example, the transformed cells can be removed from the culture medium after the culture medium reaches an OD600 of at least about 1-2 (e.g., when the transformed cells are still in the growth phase), and the transformed cells are placed in a fresh Minimum Essential Medium supplemented with phenylalanine. This optional subculture step can be performed one or more times. Optionally, when the culture contains excess tCA, for example, when the culture medium contains at least 0.5, 1.0, 1.5 or 2.0 mM tCA, a subculture step is performed to remove the transformed cells from the culture. In an eighth aspect of the present invention, the present invention provides a method for treating a disease or condition in a subject in need thereof, wherein the disease or condition is characterized by elevated levels of phenylalanine, wherein the method comprises administering to the subject a PAL variant according to the first aspect of the present invention; a conjugate according to the second aspect of the present invention; a the pharmaceutical composition described above; the polynucleotide described in the fourth aspect of the present invention; the expression vector described in the fifth aspect of the present invention and / or the bacteria and modified cells described in the sixth aspect of the present invention. According to one embodiment of the present invention, in the method for treating a disease or condition in a subject in need according to the present invention, administration is carried out by any suitable method known in the art, such as oral, percutaneous, transmucosal, intrapulmonary (comprising atomization), intramuscular, subcutaneous or intravenous administration. According to a preferred embodiment of the present invention, by subcutaneous or oral administration. According to one embodiment of the present invention, the dosage form is selected for oral or parenteral preparation in a manner known in the art. The pharmaceutical composition according to the present invention can be, for example, in the form of a solid, semi-solid or liquid dosage form, such as a tablet, suppository, pill, capsule, powder, liquid, suspension, cream, ointment, lotion or the like, such as a unit dosage form suitable for single administration of a precise dose. The pharmaceutical composition according to the present invention can include a therapeutically effective amount of a PAL variant according to the present invention in combination with a pharmaceutically acceptable carrier; in addition, other agents, adjuvants, diluents, buffers, etc. can be optionally included. According to one embodiment of the invention, a slow release or sustained release system may be employed so as to maintain a constant dosage level. According to one embodiment of the present invention, the PAL variant according to the first aspect of the present invention or the conjugate according to the second aspect of the present invention is subcutaneously administered to the subject to treat diseases and disorders characterized by elevated levels of phenylalanine, in particular, the disease or disorder is phenylketonuria (PKU). According to one embodiment of the present invention, the conjugate according to the second aspect of the present invention is subcutaneously administered to the subject, thereby increasing the half-life of the PAL variant enzyme when administered to a subject in need thereof. According to one embodiment of the present invention, the conjugate comprises a PAL variant enzyme chemically coupled to a polyethylene glycol (PEG) polymer. BRIEF DESCRIPTION OF THE DRAWINGS In order to make the above-mentioned purpose, features and advantages of the present invention easier to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to fully understand the present invention, but the present invention can also be implemented in other ways different from those described herein, so the present invention is not limited to the specific embodiments disclosed below. FIG1 shows a schematic diagram of phenylalanine ammonia lyase (PAL) or a PAL variant catalyzing phenylalanine to produce trans-cinnamic acid; FIG2A schematically shows the three-dimensional structure of a single subunit in a PAL tetramer; FIG2B schematically shows the positions of some mutation sites in the three-dimensional structure; 3A and 3B schematically show the PAL high-throughput screening sites and amino acid substitutions with phenylalanine ammonia lyase activity according to the present invention; FIG4 schematically shows the SEC-HPLC profiles of PAL and its variants (PAL201: C503L / C565P; PAL202: G218A / C503L / C565P; PAL204: M222L / C503L / C565P) after combined purification (SEC chromatography column Ultrahydrogel 1000 (Waters), mobile phase: 0.3 ml / min PBS buffer solution); FIG5 schematically shows an SDS-PAGE image of some PAL variants after affinity chromatography purification according to the present invention; FIG6 schematically shows the changes in the enzyme activity of the PAL variant C503L / C565P according to the present invention at pH values ​​between 6.5 and 9.0; FIG7 schematically shows the changes in the thermal stability of the enzyme activities of the PAL variants according to the present invention and the wild-type PAL at a temperature of 37-70° C.; FIG. 8 schematically shows the HPLC-SEC spectra of some PAL variants before and after modification with polyethylene glycol (PEG) according to the present invention. DETAILED DESCRIPTION As illustrated and exemplified in the accompanying drawings, from the following more detailed description of specific embodiments, the above and other purposes, elements and advantages will become apparent. In terms of its scope of use, the terms "one", "a kind of" and the singular form used in the claims and specification herein should be understood as including the plural form of the same word, so that these terms represent that one or more things are provided. The terms "at least one" and "one or more" can be used interchangeably. The term "single" is applied to indicate one and only one of something. Similarly, when a specific number of things are needed, other specific integer values, such as "two" will be used. The terms "preferably", "preferred", "preferably", "optionally", "can" and similar terms are used to indicate that the quoted project, condition or step are optional (that is, not required) features in the embodiment. Unless otherwise stated, the scope described as "between a and b" includes the value of "a" and "b". Example 1 Construction of genetically engineered bacteria E. coli BL21 (DE3) / pET28a-PAL The phenylalanine ammonia lyase PAL encoding gene from Anabaena variant (amino acid sequence as shown in SEQ ID NO.1) was codon optimized, and the nucleotide sequence of the codon-optimized PAL gene was shown in SEQ ID NO: 2. The nucleotide sequence of the codon-optimized PAL gene (SEQ ID NO.2) was artificially synthesized and inserted between Nco I and Xho I of pET28a to obtain the recombinant plasmid pET28a-PAL, and the electrophoresis diagram is shown in Figure 2. Take out 5μL of plasmid pET28a-PAL, add it to a 1.5ml EP tube containing 50ul E.coli BL21(DE3) competent cells, flick the tube wall to mix, and place on ice for 30min. After heat shock in a 42℃ water bath for 45s, immediately place on ice for 2min. Add 1mL LB liquid culture medium to the EP tube and culture at 37℃ for 1h. Centrifuge the culture solution at 4500rpm for 4min. Take out 800μL of the supernatant, resuspend the bacteria with the remaining supernatant, and take out 100μL to apply on LB solid culture medium containing 50μg / mL kanamycin. Culture at 37℃ for 12-14h to obtain the engineered bacteria E.coli BL21(DE3) / pET28a-PAL that recombinantly expresses PAL. LB liquid culture medium composition: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, solvent is distilled water, pH is about 7.0. Example 2 Construction of pET28a-PAL saturated mutation plasmid and expression bacteria containing the mutation plasmid Using the plasmid pET28a-PAL prepared in Example 1 as a template, a pair of primers was designed, and the mutation site primer sequence was set to "NNN". The whole plasmid was amplified using the inverse PCR technique, and the plasmid was digested with DpnI. The plasmid was purified using a kit (TaKaRa), and the plasmid was introduced into the cloning host bacteria DH5α using an electroporation method. After the plasmid was enriched by the cloning bacteria, the plasmid was extracted and then transformed into E. coli BL21 (DE3). The primers for each point saturation mutation are shown in Table 1. Table 1 Primers for site saturation mutagenesis The inverse PCR amplification system is shown in Table 2. Table 2 PCR amplification system The PCR reaction process was as follows, and the PCR products were detected by 0.8% agarose gel electrophoresis. Table 3 PCR amplification conditions Take out 5 μL of the obtained saturated mutant plasmid and add it to 50 μL of E. coli Top10 competent medium, flick the tube wall to mix, and place on ice for 30 minutes. Heat shock in a 42℃ water bath for 45 seconds, and immediately place on ice for 2 minutes. Add 1mL of LB liquid culture medium to the tube and culture at 37℃ for 1 hour. Centrifuge the culture at 4500rpm for 4 minutes and take out 800μL of the supernatant. Resuspend the bacteria with the remaining supernatant, take out 100μL and apply it on LB solid culture medium containing 100μg / mL kanamycin. 37℃, After culturing for 12-16 hours, clones containing the mutant plasmid were obtained. After enrichment culture of the clones, the plasmid was extracted and transformed into E. coli BL21 (DE3) competent cells. The above transformation steps were repeated to obtain expression bacteria containing the mutant plasmid. Example 3: High-throughput screening of PAL mutant plasmid expressing bacteria 1. Bacteria culture Pick up mutant plasmid expression bacteria (with or without His tag) from the plate, inoculate into a 96-deep-well plate with LB / Kana+ culture medium, culture at 37°C and 800 rpm for 16 h, take out 100 ul and transfer to another 96-deep-well plate, each well has 900 ul TB culture medium, Kana and IPTG have been added, culture at 37°C and 800 rpm for 16 h, then centrifuge at 3000 rpm at 4°C for 30 min, remove the supernatant, and obtain wet bacterial cells. 2. Bacterial cell disruption The wet bacterial cells in the 96-deep-well plate were frozen and thawed twice in a refrigerator at about -80°C, and then the bacterial cells were resuspended in Tris-HCl buffer (pH = 8.5) containing lysozyme, shaken at 800 rpm for 30 min at 37°C, and centrifuged under the same conditions. The supernatant was the crude enzyme solution. 3. Enzyme activity detection and enzyme activity calculation Take 10ul of crude enzyme solution, add 190ul of Tris-HCl (100mM, pH8.5) buffer containing 22.5mM phenylalanine for measurement, use the empty plasmid wells and PAL wells as the control group, and the remaining wells as the experimental group. The measurement wavelength is 290nm, the parameters are set to measure absorbance, the measurement is 10min in total, and the data is measured every 30s. Enzyme activity unit definition: At 37°C and pH 8.5, 1u mole of L-phenylalanine is deaminated and converted into trans-cinnamic acid and NH 3 Defined as 1U. The enzyme activity calculation formula is as follows: Enzyme activity (U / ml) = Δc × V1 / (t × V2) Δc: product concentration change, unit is uM. By establishing a linear relationship between product concentration and absorbance, the detected product concentration change is obtained by substitution; V1: total volume of the activity test system, in ml; t: total duration of activity measurement, in minutes; V2: Volume of enzyme solution added, in ml 4. Comparison of enzyme activity between PAL variant enzyme and wild-type PAL enzyme The comparison results are shown in Table 4, where "+" indicates that the enzyme activity of the PAL variant is higher than that of the wild-type PAL enzyme, "o" indicates that the enzyme activity is equal to that of the wild-type PAL enzyme, and "-" indicates that the enzyme activity is lower than that of the wild-type PAL enzyme. Table 4 Comparison of enzyme activities of PAL variant enzymes and wild-type PAL enzymes Example 4: pET28a-PAL and its mutant plasmid expression bacterial cell culture and protein induction After pET28a-PAL and its mutant plasmids were transformed into E.coli BL21, wild-type PAL and PAL variants were obtained. Pick a single colony from the plate into LB / Kana liquid medium, culture it overnight at 37°C at 200rpm for 12-16h, transfer the bacterial solution to new LB / Kana liquid medium with an inoculation ratio of 2%, culture it at 37°C at 200rpm for 1-2h, when OD600 is between 0.6-0.8, add IPTG with a final concentration of 0.2mM, and induce it at 30°C at 200rpm for 12-16h. After induction, the bacteria were collected by centrifugation at 6000 rpm for 10 min at 4°C. The wet bacteria were stored at -80°C. Example 5: Protein specific activity determination Protein concentration detection: The protein measurement method refers to the method of BCA test kit (Thermo), which is incorporated herein by reference; wherein the linear range is 25ug / ml to 2000ug / ml. This embodiment uses a 96-well test plate, and the volumes of the added enzyme solution and test solution are 10ul and 200ul, or 25ul and 200ul, respectively. Enzyme activity detection: The enzyme activity of wild-type PAL and PAL variants was detected using the enzyme activity detection method described in Example 3. Specific activity calculation: Specific activity (U / mg) = enzyme activity (U / ml) / protein concentration (mg / ml) Example 6: Purification of wild-type PAL and PAL variants Cell lysis: The frozen cell pellet was thawed and resuspended in a buffer of 20 mM Tris-HCl, 100 mM NaCl at pH 8.0 at room temperature to form a cell slurry with a density of about 120 to 140 OD600. The cells were passed twice through a Niro NS30006 homogenizer at 700-800 Bar (where the temperature was controlled below 30°C) to lyse the cells by homogenization. After homogenization, the pH was adjusted to about 8.0 by adding 1N NaOH to the lysate. The cell lysate was gradually heated to 55°C, maintained at 55°C for 30 to 120 minutes, and then cooled. The cell lysate was clarified by centrifugation. Ammonium sulfate was added to the supernatant of the lysate containing the target enzyme to a saturation of 30-70% and allowed to stand overnight. The precipitate was collected by centrifugation. The enzyme precipitate was resuspended in a Tris buffer solution at pH 8.5. Protein purification: 1. PAL and its variants containing His tag: The Ni-NTA affinity chromatography column was equilibrated with 5 mM imidazole (50 mM Tris-HCl, pH 8.5, 0.5 M NaCl) for 2-5 column volumes; the cell lysate filtered through a 0.45 μm filter membrane or the above-mentioned Tris resuspension was loaded into the chromatography column; the elution was performed with a gradient of 5 mM-200 mM imidazole (50 mM Tris-HCl, pH 8.5, 0.5 M NaCl) buffer solution, and the elution peak was collected. The molecular weight and purity of the fusion protein were detected by SDS-PAGE. The results of some PAL variants are shown in FIG5 , wherein FIG5 shows a total of 9 lanes, wherein from left to right are protein standard Marker (lane 1) and PAL variants containing His tags (lanes 2 to 9), wherein lanes 2 to 9 represent: PAL variants containing C503L / C565P mutations, PAL variants containing mutations T102R / C503L / C565P, PAL variants containing G218A / C503L / C565P mutations, PAL variants containing G218S / C503L / C565P mutations, PAL variants containing M222L / C503L / C565P mutations, PAL variants containing S438N / C503L / C565P mutations, PAL variants containing I439V / C503L / C565P mutations; the standard sample bands shown are 200, 140, 95, 65, 52, 41, 33, 25, 17, 10 from top to bottom. It can be seen that the target PAL variant appears between the relative molecular weight of 52-65 kDa; 2. PAL and its variants without His tag: The Tris resuspended enzyme solution is sequentially passed through a hydrophobic interaction (HIC) column and an anion exchange (AIEX) column to purify the PAL variant. It should be noted that the order of use of the hydrophobic interaction (HIC) column and the anion exchange (AIEX) column can be exchanged, or only one chromatographic column can be used as a purification tool. It should be understood that other AIEX and HIC column resins can be used, and the HIC column can be replaced by size exclusion chromatography. The precipitated resuspended enzyme solution is diluted to 2 times with a 50mM Tris-HCl buffer solution with a pH value of 8.5, and loaded into a TPGQ AIEX column balanced with a 50mM Tris-HCl buffer solution with a pH value of 8.5. The column is rinsed with a 50mM Tris-HCl, 400mM NaCl buffer solution with a pH value of 8.5, and PAL is eluted with a gradient of a Tris-HCl buffer (50mM, pH 8.5) containing 0 to 1000mM NaCl. The components with enzyme activity are collected, ultrafiltered and concentrated. Hydrophobic column chromatography: A HIC column (t-Butyl, Tosoh) was charged with 1 M (NH 4 ) 2SO 4 The PAL enzyme solution was pre-equilibrated with a Tris-HCl buffer solution (50 mM, pH 8.5). 4 ) 2 SO 4 The solution was diluted 2-fold with Tris-HCl buffer solution (50 mM, pH 8.5) and loaded into the HIC column. 4 ) 2 SO 4 The protein was gradient eluted with a Tris-HCl buffer solution (50 mM, pH 8.5), and the fractions with enzyme activity were collected and desalted by ultrafiltration concentration. The specific activity data of the wild-type PAL enzyme and some PAL variant enzymes after purification are summarized in Table 5. Table 5. Specific activities of wild-type PAL enzyme and PAL variant enzyme Example 7: Comparison of Aggregation of Wild-Type PAL Enzyme and PAL Variant Enzyme The purified PAL was concentrated and the concentrated protein solution (2.5 mg / ml) was incubated at 37°C for 2 hours to accelerate the aggregation of the purified PAL protein in solution. The PAL protein was separated by SEC-HPLC to detect aggregation. To determine whether disulfide cross-linking was the cause of aggregation, 50 mM dithiothreitol (DTT) was added to the concentrated protein solution, followed by incubation at 37°C for 2 h. For the aggregation-prone wild-type PAL solution, the solution was further concentrated to about 10 mg / mL by ultrafiltration and incubated for 2 hours at 37° C. For the aggregation-resistant PAL variant, the solution was further concentrated to about 30 mg / mL and incubated for 2 hours at 37° C. As shown in Table 6, the purified wild-type PAL enzyme aggregated after incubation at 37°C for 2 hours. As expected, this aggregation was exacerbated when the PAL protein was concentrated before incubation at 37°C for 2 hours. Exposure of the concentrated protein to DTT prevented aggregation, indicating that aggregation was caused by disulfide cross-linking. In contrast, the purified PAL variant (C503S / C565P) and PAL variant (C503S / C565L) did not form aggregates after incubation at 37°C for 2 hours, indicating that the PAL variants have lower aggregation than the wild-type PAL enzyme. Table 6. Effect of different treatments of PAL variants on aggregate formation “-” indicates no aggregation, “+” indicates aggregation, and “++” indicates severe aggregation. Example 8: pH adaptation curves of wild-type PAL enzyme and PAL variant enzyme The optimal pH values ​​of the wild-type PAL enzyme and the selected PAL variants were determined by the specific activity assay described in Example 5. 10ul of pure enzyme solution (total protein 0.2μg) + 190ul of 100mM Tris-HCl (pH8.5) buffer containing 22.5mM Phe was taken for assay, and the enzyme activity was detected in a 200μL system at 37°C. The pH values ​​of 100 mM Tris-HCl (pH 8.5) buffer containing 22.5 mM Phe were set to 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0, respectively. The measurement results are summarized in Figure 6, where all results are normalized to the enzyme activity of the PAL variant with C503L / C565P mutation at pH 7.5. As can be seen from Figure 6, the PAL variant with C503L / C565P mutation has the best enzyme activity in the pH range of 7.5 to 8.5. Example 9: Temperature stability of wild-type PAL enzyme and PAL variant enzyme The effect of temperature on the stability of wild-type PAL enzyme and selected PAL variant enzymes was determined by incubating the proteins in 50 mM Tris-HCl, pH 7.5 for 2 hours at a temperature range of 37°C to 70°C and then measuring the enzyme activity. Each enzyme reaction was performed with 1 μg of PAL protein and 22.5 mM phenylalanine as substrates in a total reaction volume of 200 μL at 37°C. The measurement results are summarized in Figure 7. All results were normalized to the enzyme activity of the PAL variant with M222L / C503L / C565P mutations at 37°C. Example 10: PEG-modified PAL variants The solution containing PAL protein was dialyzed in pH=8.0, 10mM PBS buffer to fix the final protein concentration at 2.5mg / ml. PEG (PEG-NHS) with a molecular weight of 20kDa was added to the above PAL solution to fix the final PEG concentration at 75mg / ml. The reaction solution of PEG and PAL was reacted at room temperature for 2-4 hours, or at 4°C overnight. When the reaction was terminated, 10μl of the above reaction solution was immediately taken for dilution, and the enzyme activity was determined according to the method in Example 5, and the change in enzyme activity before and after the reaction of PAL and PEG was calculated (see Table 7). The reaction solution containing the PEG-PAL conjugate was placed in a dialysis bag (molecular weight cutoff 300kD) and fully dialyzed in PBS 7.4 buffer solution. The final product was detected by HPLC-SEC. As shown in Figure 8, after the three PAL variants were modified with PEG, they all showed a significant left shift in the HPLC-SEC spectrum, indicating that the PAL variants were successfully conjugated with PEG and the overall molecular size was significantly increased. Table 7. Changes in enzyme activity of PAL variants after PEG modification It is worth noting that in embodiments according to the present invention, the steps of the method can be performed in any suitable order, unless otherwise specified herein or clearly contradictory to the context. Unless otherwise specified herein or clearly contradictory to the context, these steps can be repeated or repeated any number of times to achieve the desired goal. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present invention and is not intended to limit the scope of the present invention unless otherwise required. No language in the specification should be construed as indicating that any non-claimed element is essential to the practice of the present invention. Preferred aspects of the present invention are described herein, including the best mode known to the inventor for carrying out the present invention. After reading the foregoing description, variations of these preferred aspects may become apparent to those of ordinary skill in the art. The inventor expects those of ordinary skill in the art to adopt such variations where appropriate, and the inventor intends that the present invention be implemented in situations other than those specifically described herein. Although various improvements have been described herein with reference to specific embodiments of the invention, it should be understood that such description is by way of illustration only and should not be construed as limiting the scope of any claimed invention. Therefore, the scope and content of any claimed invention will be limited only by the terms of the appended claims in the current form or as modified during the examination or in the form implemented in any continuation application. In addition, it should be understood that, unless otherwise stated, the features of any specific embodiment discussed herein may be combined with one or more features of any one or more embodiments discussed or considered in other ways herein. Those of ordinary skill in the art will recognize that, without departing from the spirit and scope of the present application as described in the appended claims, some modifications and changes may be made to the described embodiments, and these modifications and changes fall within the scope of protection of the present invention.

Claims

1. A variant of phenylalanine ammonia lyase (PAL), the phenylalanine ammonia lyase (PAL) being derived from Anabaena variabilis and comprising the amino acid sequence shown in SEQ ID NO: 1, wherein In, the variant comprises: Amino acid substitution C503S / C565L, such that the variant comprises the amino acid sequence shown in SEQ ID NO: 2; or Amino acid substitution C503S / C565P, such that the variant comprises the amino acid sequence shown in SEQ ID NO: 3; or Amino acid substitution C503L / C565P, so that the variant comprises the amino acid sequence shown in SEQ ID NO:4 。 2. The variant of phenylalanine ammonia lyase according to claim 1, characterized in that The variant further comprises one or more of the following amino acid substitution point mutations: T102R, T102H, I165L, I165V, G166A, G166S, G218A, G218S, G218N, M222L, M222I, N437S, S438N, S438A, I439V, I439C, I439A, I439K, A440T, A440G, A440V, A440T, R441N, R442Q, T445V, T445S, A447S, E448K, N453G, Q457L, and G45 8K。 3. The variant of phenylalanine ammonia lyase according to claim 1, Characterized in that the variant comprises: Amino acid substitution C503L / C565P / 222L, or amino acid substitution C503L / C565P / G 218A。 4. The variant of phenylalanine ammonia lyase according to claim 1 or 2 The variant is characterized in that, in the reaction of converting phenylalanine into trans-cinnamic acid, the activity of the variant is higher than that of the phenylalanine comprising the amino acid sequence shown in SEQ ID NO:

1. Alanine ammonia lyase.

5. The phenylalanine ammonia lyase according to claim 1 or 2 A variant, characterized in that the variant exhibits the same stability at a temperature ranging from 37°C to 70°C as compared to the phenylalanine ammonia lyase comprising the amino acid sequence shown in SEQ ID NO:

1. or greater stability.

6. The phenylalanine solution according to claim 1 or 2 A variant of an aminoacidase, characterized in that the variant is different from a phenylalanine hydrolase comprising the amino acid sequence shown in SEQ ID NO:

1. It has low aggregation compared to ammonia enzyme.

7. A conjugate, characterized in that the conjugate comprises a variant of phenylalanine ammonia lyase according to claim 1 or 2 and a polymer, wherein The polymer is a water-soluble polymer.

8. According to the rights The conjugate according to claim 7 is characterized in that the water-soluble polymer is polyethylene glycol, polyzwitterionic polymer and polyoxazoline Any one or a mixture of two or more Compound.

9. A pharmaceutical composition comprising: (i) Phenylalanine ammonia lyase according to claim 1 or 2 Change or the conjugate according to claim 6; and (ii) a suitable pharmaceutically acceptable carrier selected from Tris-HCl, NaCl, L-phenylalanine (L-Phe), glycine (Gly) and trans-cinnamon Any one or a mixture of two or more acids.

10. The pharmaceutical composition according to claim 9, characterized in that , the pharmaceutical composition can be used to treat phenylketonuria.

11. For encoding a method according to any one of claims 1 to 6. Polynucleotides of variants of phenylalanine ammonia lyase Acid, characterized in that the polynucleotide is DNA or RNA 。 12. The polynucleotide according to claim 11, wherein the polynucleotide is codon optimized so that Polynucleotides useful for expressing phenylalanine ammonia lyase in Escherichia coli Variant of .

13. The polynucleotide according to claim 11, wherein the polynucleotide is codon optimized so that the polynucleotide Acid can be used in animal cells, especially human A variant of phenylalanine ammonia lyase is expressed in the cells.

14. An expression vector, characterized in that the expression vector comprises the 3, wherein the expression vector Can be expressed in living cells.

15. The expression vector according to claim 14, wherein The feature is that the expression vector is a virus, and the virus includes an adenovirus viruses, adeno-associated viruses, and lentiviruses.

16. The expression vector according to claim 14, characterized in that the expression vector is a plasmid comprising a promoter The promoter is according to any one of claims 11 to 13 One of the polynucleotides is operably linked.

17. According to the rights The expression vector according to claim 16 , characterized in that the plasmid is transfected into the cell via liposomes or cationic polymers.

18. A bacterium, characterized in that the bacterium has integrated into its genome the gene according to claim The polynucleotide described in 11 and / or capable of expressing the phenylalanine ammonia hydrolysis according to any one of claims 1 to 6 Enzyme variants.

19. A modified cell comprising: According to rights The variant of the phenylalanine ammonia lyase according to any one of claims 1 to 6; root The polynucleoside according to any one of claims 11 to 13 acid; A table according to any one of claims 14 to 17 to the carrier; and Optionally, the Modified cells are modified animal cells.

20. According to claim 19 The modified cells are characterized in that they are human cells.

21. A method for preparing a variant of phenylalanine ammonia lyase, characterized in that the method comprises culturing the bacterium according to claim 18 or the bacterium according to claim 1 in a culture medium.

9. The modified cell of claim 9 to produce a variant of phenylalanine ammonia lyase and to separate the variant of phenylalanine ammonia lyase from the modified cell and / or the culture medium 。 22. A variant of phenylalanine ammonia lyase according to any one of claims 1 to 6 for use in the preparation of a method for treating an individual with elevated phenylalanine levels. High in drug use.

23. A variant of phenylalanine ammonia lyase according to any one of claims 1 to 6 for use in the preparation of a preparation for treating phenylketonuria Use in medicines for the treatment of diseases.

24. A method of treating a disease or condition in a subject in need thereof, wherein the disease or condition The disease is characterized by elevated levels of the amino acid phenylalanine. Characterized in that the method comprises administering to the subject a variant of phenylalanine ammonia lyase according to any one of claims 1 to 6; a conjugate according to claim 7 or 8; The pharmaceutical composition according to claim 9 or 10 or the multicore according to any one of claims 11 to 13 nucleotide; an expression vector according to any one of claims 14 to 17; a bacterium according to claim 18, and / or a modified gene according to claim 19 or 20 Decorative cells.

25. The method according to claim 24 The method, wherein the subject is subcutaneously administered The variant of any one of claims 1 to 6 or the conjugate of claim 7 or 8.

26. The method according to claim 24, Characterized in that the disease or disorder is phenylketonuria.

27. A variant of the phenylalanine ammonia lyase according to any one of claims 1 to 6, wherein the variant is expressible in plants, fungi and bacteria.

28. According to any one of claims 1 to 6 The variant of the phenylalanine ammonia lyase described in item 1, wherein the variant can be expressed in Nostoc punctata, Anabaena variant, Rhodosporidium toruloides, Streptomyces marineus, Anastrozole nidulans, Photorhabditis luminescens, Streptomyces verticillata, and Escherichia coli.

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