L-aspartic acid-alpha-decarboxylase mutant, nucleic acid molecule, recombinant expression vector, vector cell, production method and application

Abstract

The invention discloses an L-aspartic acid-alpha-decarboxylase mutant, a nucleic acid molecule, a recombinant expression vector, a vector cell, a production method and application. Compared with the amino acid sequence of the L-aspartic acid-alpha-decarboxylase as shown in SEQ ID No.1, the provided L-aspartic acid-alpha-decarboxylase mutant comprises amino acid mutation of one or more of the following sites: the 65th site, the 70th site, the 71th site, the 87th site, the 90th site, the 110th site, the 128 site, the 148th site, the 203th site, the 209th site, the 228th site, the 229th site, the 254th site, the 284th site, the 286th site, the 291,294 site, the 347th site, the 371,392 site and the 463th site. The L-aspartic acid-alpha-decarboxylase mutant provided by the invention is obtained by carrying out site-specific mutagenesis on the basis of insect source L-aspartic acid-alpha-decarboxylase. The L-aspartic acid-alpha-decarboxylase mutant provided by the invention is applied to preparation of beta-alanine, and has relatively high industrial application potential.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering technology and biocatalysis, and relates to an L-aspartate-alpha-decarboxylase mutant, a nucleic acid molecule, a recombinant expression vector, a vector cell, a production method and an application. Background Art

[0002] β-Alanine is widely used in medicine, chemical industry, food, environment and other fields, especially in the synthesis of calcium pantothenate, carnosine and acrylamide. It is an important industrial synthetic raw material.

[0003] In the industrial production process, β-alanine can be produced by chemical method and enzymatic method. The chemical method has disadvantages such as safety issues, high equipment cost and environmental pollution. The enzymatic method is relatively greener and more environmentally friendly, which is in line with the direction of industrial development. In the enzymatic method, L-aspartate-α-decarboxylase (ADC) is often used to catalyze the production of β-alanine.

[0004] At present, ADC is mainly divided into two types. One uses pyridoxal phosphate as a cofactor, which is mainly found in some archaea and insects; the other uses pyruvate as a cofactor, which is found in most bacteria and archaea. Among them, ADCs derived from prokaryotes need to undergo self-cleavage modification to obtain catalytic function, and have the disadvantage of irreversible inactivation based on the mechanism, which is not suitable for industrial application. ADCs derived from eukaryotes do not have mechanism inactivation and have great potential for industrial application. However, ADC enzymes derived from eukaryotes usually have low enzyme activity during heterologous expression, and there are difficulties such as harsh culture conditions when scaling up fermentation culture, which limits their industrial application. Summary of the invention

[0005] Based on this, it is necessary to provide an L-aspartate-α-decarboxylase mutant, a nucleic acid molecule, a recombinant expression vector, a vector cell, a production method and an application to meet the needs of industrial production.

[0006] In some embodiments, a L-aspartate-α-decarboxylase mutant is provided, wherein the L-aspartate-α-decarboxylase mutant comprises an amino acid mutation at one or more of the following sites compared to the amino acid sequence of the L-aspartate-α-decarboxylase shown in SEQ ID No. 1:

[0007] No. 65, No. 70, No. 71, No. 87, No. 90, No. 110, No. 124, No. 128, No. 148, No. 203, No. 204, No. 209, No. 228, No. 229, No. 254, No. 284, No. 286, No. 291, No. 294, No. 347, No. 371, No. 392 and No. 463.

[0008] In some embodiments, the provided L-aspartate-α-decarboxylase mutant comprises an amino acid mutation at one or more of the following sites:

[0009] No. 65, No. 70, No. 203, No. 204, No. 209, No. 254, No. 286, No. 291, No. 294, No. 347, No. 371, No. 392 and No. 463.

[0010] In some embodiments, the provided L-aspartate-α-decarboxylase mutant comprises one or any suitable plurality of mutations as follows:

[0011] (1) Glycine at position 65 was mutated to asparagine,

[0012] (2) Glycine at position 65 mutated to serine,

[0013] (3) Alanine at position 70 mutated to aspartic acid,

[0014] (4) Alanine at position 70 mutated to glutamic acid,

[0015] (5) Alanine at position 70 mutated to glycine,

[0016] (6) Alanine at position 70 mutated to serine,

[0017] (7) Threonine at position 71 mutated to valine,

[0018] (8) Threonine at position 71 mutated to phenylalanine,

[0019] (9) Threonine at position 71 mutated to asparagine,

[0020] (10) Threonine at position 71 mutated to arginine,

[0021] (11) Lysine at position 87 mutated to histidine,

[0022] (12) Isoleucine at position 90 mutated to phenylalanine,

[0023] (13) Alanine at position 110 mutated to glutamine,

[0024] (14) Methionine at position 124 mutated to leucine,

[0025] (15) Phenylalanine at position 128 mutated to threonine,

[0026] (16) Phenylalanine at position 128 mutated to alanine,

[0027] (17) The serine at position 148 was mutated to alanine,

[0028] (18) The serine at position 148 was mutated to glycine,

[0029] (19) The histidine at position 203 was mutated to glutamine,

[0030] (20) Methionine at position 204 was mutated to asparagine,

[0031] (21) Methionine at position 204 mutated to arginine,

[0032] (22) Methionine at position 204 mutated to valine,

[0033] (23) Threonine at position 209 mutated to serine,

[0034] (24) Threonine at position 209 mutated to tyrosine,

[0035] (25) Threonine at position 209 mutated to glutamic acid,

[0036] (26) Tyrosine at position 228 mutated to valine,

[0037] (27) The serine at position 229 was mutated to alanine,

[0038] (28) Lysine 254 was mutated to methionine,

[0039] (29) Lysine 254 was mutated to glycine,

[0040] (30) Threonine at position 284 was mutated to alanine,

[0041] (31) Threonine at position 284 mutated to phenylalanine,

[0042] (32) Threonine at position 284 mutated to tyrosine,

[0043] (33) Isoleucine at position 286 was mutated to glutamine.

[0044] (34) Proline 291 mutated to arginine,

[0045] (35) Glutamine at position 294 mutated to serine,

[0046] (36) Glutamine at position 347 mutated to isoleucine,

[0047] (37) Glutamine at position 347 mutated to leucine,

[0048] (38) Glutamine at position 347 mutated to valine,

[0049] (39) Leucine at position 371 mutated to valine,

[0050] (40) Leucine at position 371 mutated to isoleucine,

[0051] (41) Leucine at position 371 mutated to phenylalanine,

[0052] (42) Leucine at position 371 mutated to tyrosine,

[0053] (43) Leucine at position 371 mutated to aspartic acid,

[0054] (44) Glycine at position 392 mutated to isoleucine,

[0055] (45) Glycine at position 392 was mutated to valine,

[0056] (46) Glycine 392 mutated to phenylalanine, and

[0057] (47) Aspartic acid at position 463 was mutated to valine.

[0058] In some embodiments, a nucleic acid molecule is provided, wherein the nucleic acid molecule encodes the L-aspartate-α-decarboxylase mutant.

[0059] In some embodiments, a recombinant expression vector is provided, which comprises the nucleic acid molecule; optionally, the recombinant expression vector comprises a plasmid.

[0060] In some embodiments, a vector cell is provided, comprising one or more of the nucleic acid molecules or the recombinant expression vectors.

[0061] In some embodiments, the vector cells provided include E. coli cells.

[0062] In some embodiments, a method for producing an L-aspartate-α-decarboxylase mutant is provided, comprising the following steps: culturing the vector cell; and isolating the L-aspartate-α-decarboxylase mutant from the obtained culture.

[0063] In some embodiments, there is provided use of the L-aspartate-α-decarboxylase mutant or the vector cell in the preparation of β-alanine.

[0064] In some embodiments, a method for preparing β-alanine is provided, comprising the following steps: using the L-aspartate-α-decarboxylase mutant or the culture of the carrier cell to catalyze a substrate to prepare β-alanine.

[0065] The L-aspartate-α-decarboxylase mutant provided above is obtained by site-directed mutagenesis of insect-derived L-aspartate-α-decarboxylase. The provided L-aspartate-α-decarboxylase mutant has high enzyme activity, can be used to prepare β-alanine, and has high catalytic efficiency. DETAILED DESCRIPTION

[0066] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0068] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0069] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0070] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0071] In this application, the "suitable" mentioned in "suitable combination", "suitable method", "any suitable method", etc. is based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0072] In the present application, "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present application.

[0073] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0074] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0075] In the present invention, in the "first aspect", "second aspect", "third aspect", "fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0076] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0077] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0078] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0079] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0080] The "room temperature" in this application generally refers to 5°C to 30°C, preferably 25±5°C.

[0081] In the present application, the term "mutation" refers to the deletion, addition or substitution of amino acid residues in the amino acid sequence of a protein or polypeptide compared to the amino acid sequence of a reference protein or polypeptide. Throughout the specification and claims, the substitution of an amino acid at a specific position in a protein sequence is annotated with, for example, G65N, where the annotation G65N refers to the mutation of the glycine (G) residue at position 65 of the amino acid sequence of the reference protein to an asparagine (N) residue, G65S refers to the mutation of the glycine (G) residue at position 65 of the amino acid sequence of the reference protein to a serine (S) residue, A70D refers to the mutation of the alanine (A) residue at position 70 of the amino acid sequence of the reference protein to an aspartic acid (D) residue, A70E refers to the mutation of the alanine (A) residue at position 70 of the amino acid sequence of the reference protein to a glutamic acid (E) residue, and A70G refers to the mutation of the alanine (A) residue at position 70 of the amino acid sequence of the reference protein to a glutamic acid (E) residue. The 71st threonine (T) residue in the amino acid sequence of the reference protein is mutated into a phenylalanine (F) residue. The 71st threonine (T) residue in the amino acid sequence of the reference protein is mutated into an asparagine (N) residue. The 71st threonine (T) residue in the amino acid sequence of the reference protein is mutated into an arginine (R) residue. The 71st threonine (T) residue in the amino acid sequence of the reference protein is mutated into an arginine (R) residue. The 71st threonine (T) residue in the amino acid sequence of the reference protein is mutated into an arginine (R) residue. The 71st threonine (T) residue in the amino acid sequence of the reference protein is mutated into an arginine (R) residue. The 71st threonine (T) residue in the amino acid sequence of the reference protein is mutated into an arginine (R) residue. The 87th lysine (K) residue of the amino acid sequence of the reference protein is mutated to a histidine (H) residue, I90F refers to the 90th isoleucine (I) residue of the amino acid sequence of the reference protein is mutated to a phenylalanine (F) residue, A110Q refers to the 110th alanine (A) residue of the amino acid sequence of the reference protein is mutated to a glutamine (Q) residue, M124L refers to the 124th methionine (M) residue of the amino acid sequence of the reference protein is mutated to a leucine (L) residue, F128T refers to the 128th phenylalanine (F) residue of the amino acid sequence of the reference protein is mutated to a threonine (T) residue, and F128A refers to the 128th phenylalanine ( F) residue mutates to an alanine (A) residue, S148A refers to the mutation of the 148th serine (S) residue of the amino acid sequence of the reference protein to an alanine (A) residue, S148G refers to the mutation of the 148th serine (S) residue of the amino acid sequence of the reference protein to a glycine (G) residue, H203Q refers to the mutation of the 203rd histidine (H) residue of the amino acid sequence of the reference protein to a glutamine (Q) residue, M204N refers to the mutation of the 204th methionine (M) residue of the amino acid sequence of the reference protein to an asparagine (N) residue, and M204R refers to the mutation of the 204th methionine (M) residue of the amino acid sequence of the reference protein to an arginine (R) residue,M204V refers to the mutation of the methionine (M) residue at position 204 of the amino acid sequence of the reference protein to a valine (V) residue, T209S refers to the mutation of the threonine (T) residue at position 209 of the amino acid sequence of the reference protein to a serine (S) residue, T209Y refers to the mutation of the threonine (T) residue at position 209 of the amino acid sequence of the reference protein to a tyrosine (Y) residue, T209E refers to the mutation of the threonine (T) residue at position 209 of the amino acid sequence of the reference protein to a glutamic acid (E) residue, Y228V refers to the mutation of the tyrosine (Y) residue at position 228 of the amino acid sequence of the reference protein to a valine (V) residue, S229A refers to the mutation of the tyrosine (Y) residue at position 229 of the amino acid sequence of the reference protein The serine (S) residue mutates to an alanine (A) residue, K254M refers to the mutation of the lysine (K) residue at position 254 of the amino acid sequence of the reference protein to a methionine (M) residue, K254G refers to the mutation of the lysine (K) residue at position 254 of the amino acid sequence of the reference protein to a glycine (G) residue, T284A refers to the mutation of the threonine (T) residue at position 284 of the amino acid sequence of the reference protein to an alanine (A) residue, T284F refers to the mutation of the threonine (T) residue at position 284 of the amino acid sequence of the reference protein to a phenylalanine (F) residue, T284Y refers to the mutation of the threonine (T) residue at position 284 of the amino acid sequence of the reference protein to a tyrosine (Y) residue, and I284A refers to the mutation of the threonine (T) residue at position 284 of the amino acid sequence of the reference protein to an alanine (A) residue. 6Q refers to the mutation of the 286th isoleucine (I) residue in the amino acid sequence of the reference protein to a glutamine (Q) residue, P291R refers to the mutation of the 291st proline (P) residue in the amino acid sequence of the reference protein to an arginine (R) residue, Q294S refers to the mutation of the 294th glutamine (Q) residue in the amino acid sequence of the reference protein to a serine (S) residue, Q347I refers to the mutation of the 347th glutamine (Q) residue in the amino acid sequence of the reference protein to an isoleucine (I) residue, Q347L refers to the mutation of the 347th glutamine (Q) residue in the amino acid sequence of the reference protein to a leucine (L) residue, and Q347V refers to the mutation of the 347th L371V refers to the mutation of the 371st leucine (L) residue in the amino acid sequence of the reference protein to a valine (V) residue, L371I refers to the mutation of the 371st leucine (L) residue in the amino acid sequence of the reference protein to an isoleucine (I) residue, L371F refers to the mutation of the 371st leucine (L) residue in the amino acid sequence of the reference protein to a phenylalanine (F) residue, L371Y refers to the mutation of the 371st leucine (L) residue in the amino acid sequence of the reference protein to a tyrosine (Y) residue, and L371D refers to the mutation of the 371st leucine (L) residue in the amino acid sequence of the reference protein to an aspartic acid (D) residue.G392I refers to the mutation of the glycine (G) residue at position 392 of the amino acid sequence of the reference protein to an isoleucine (I) residue, G392V refers to the mutation of the glycine (G) residue at position 392 of the amino acid sequence of the reference protein to a valine (V) residue, G392F refers to the mutation of the glycine (G) residue at position 392 of the amino acid sequence of the reference protein to a phenylalanine (F) residue, and D463V refers to the mutation of the aspartic acid (D) residue at position 463 of the amino acid sequence of the reference protein to a valine (V) residue.

[0082] In this application, the term "mutant" refers to a polynucleotide or polypeptide that contains changes (i.e., substitutions, insertions and / or deletions) at one or more (e.g., several) positions relative to a wild-type or compared polynucleotide or polypeptide, wherein substitution refers to replacing a nucleotide or amino acid occupying a position with a different nucleotide or amino acid. Deletion refers to the removal of a nucleotide or amino acid occupying a position. Insertion refers to the addition of a nucleotide or amino acid adjacent to and immediately following the nucleotide or amino acid occupying the position.

[0083] In this application, the term "vector" refers to a nucleic acid molecule capable of transporting or transferring an external nucleic acid molecule. The term encompasses both expression vectors and transcription vectors. The term "expression vector" refers to a vector capable of expressing an insert in a target cell and generally containing control sequences (such as enhancers, promoters, and terminator sequences) that drive the expression of the insert. The term "transcription vector" refers to a vector that can be transcribed but not translated. Transcription vectors are used to amplify their inserts. External nucleic acid molecules are referred to as "inserts" or "transgenics". Vectors are generally composed of inserts and larger sequences used as the backbone of the vector. Based on the structure or source of the vector, the main types of vectors include plasmid vectors, cosmid vectors, phage vectors (such as lambda phage), viral vectors (such as adenovirus vectors), and artificial chromosomes.

[0084] In the present application, the term "plasmid" refers to an extrachromosomal element that often carries genes that are not part of the core metabolic machinery of the cell, and is typically in the form of a circular double-stranded DNA molecule. These elements can be autonomous replication sequences, genome integration sequences, phage or nucleotide sequences from any source, linear, circular or supercoiled single-stranded or double-stranded DNA or RNA. Typically, a plasmid contains a functional origin of replication in a host cell (e.g., Escherichia coli), and a selectable marker for detecting host cells containing the plasmid. In some embodiments, a plasmid is a closed-loop DNA molecule. A "co-expression plasmid" refers to different types of plasmids that can be expressed in the same host bacteria.

[0085] In this application, the term "nucleic acid" refers to any linear or sequentially arranged nucleotides and nucleosides, for example, cDNA, genomic DNA, mRNA, tRNA, oligonucleotides, oligonucleosides and derivatives thereof. Nucleic acids may include bacterial plasmid vectors, including expression, cloning, cosmid and transformation vectors, and nucleic acids may include modified or derived nucleotides and nucleosides.

[0086] In this application, the term "nucleotide sequence" refers to oligonucleotides, nucleotides or polynucleotides and fragments or portions thereof, and refers to DNA or RNA of genomic or synthetic origin, which may be single-stranded or double-stranded, and represents a sense or antisense strand. The terms "polynucleotide", "oligonucleotide", "nucleotide sequence" and "nucleic acid" are used interchangeably herein and include, but are not limited to, coding sequences. That is, one or more polynucleotides or one or more nucleic acid sequences that are transcribed and translated into polypeptides in vitro or in vivo when placed under the control of appropriate regulatory or control sequences; control sequences, for example, translation start and stop codons, promoter sequences, ribosome binding sites, polyadenylation signals, transcription factor binding sites, transcription termination sequences, upstream and downstream regulatory domains, enhancers, silencers, DNA sequences to which one or more transcription factors bind and positively (induce) or negatively (inhibit) alter the promoter activity of a gene, etc.

[0087] In this application, the term "expression" refers to the conversion of sequence information into corresponding expression products, including direct transcription products (such as mRNA, tRNA, rRNA, antisense RNA, ribozymes, structural RNA or any other type of RNA) or proteins produced by mRNA translation.

[0088] In this application, the term "coding" refers to a DNA polynucleotide sequence that can be transcribed into RNA (mRNA) that translates protein; or can be transcribed into RNA that does not translate protein (non-coding RNA such as tRNA and rRNA); or can be translated into an RNA polynucleotide sequence that can be translated into protein.

[0089] In this application, the terms "DNA", "RNA" or "nucleic acid" or "nucleic acid fragment" or "polynucleotide" refer to any one or more nucleic acid segments present in a polynucleotide or construct. Nucleic acids or fragments thereof can be provided in linear (e.g., mRNA) or circular (e.g., plasmid) form, as well as in double-stranded or single-stranded form. An "isolated" nucleic acid or polynucleotide refers to a nucleic acid molecule, DNA or RNA, that has been separated from its natural environment. For example, in the context of the present invention, a recombinant polynucleotide contained in the vector is isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified polynucleotides in solution, etc.

[0090] In the present application, "gene" refers to a polynucleotide comprising nucleotides encoding functional molecules, including functional molecules (biologically active RNA) produced only by transcription or functional molecules (such as polypeptides) produced by transcription and translation. The term "gene" includes cDNA and genomic DNA nucleic acids, and also refers to a nucleic acid fragment expressing a specific RNA, protein or polypeptide, which contains regulatory sequences before (5' non-coding sequence) and after (3' non-coding sequence) the coding sequence. "Natural gene" refers to any gene found in nature with its own regulatory sequence. "Chimeric gene" refers to any gene that is a non-natural gene and contains non-natural co-existing regulatory and / or coding sequences. "Endogenous gene" refers to a natural gene located in its natural position in the genome of an organism. "Foreign gene" or "heterologous gene" refers to a gene that is not usually in the host and is introduced into the host by gene transfer.

[0091] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to a polymer of amino acid residues.

[0092] In this application, the term "recombinant" nucleic acid refers to non-natural nucleic acid, for example, by artificial intervention to connect originally separate nucleic acid fragments together to produce a combined nucleic acid fragment with a desired function. Artificial combination is often accomplished by chemical synthesis or by artificial manipulation of nucleic acid segments (e.g., by genetic engineering technology).

[0093] In the present application, the term "introduced" or "introduced into" a cell refers to the transformation of a nucleic acid molecule (e.g., a plasmid, a linear nucleic acid fragment, RNA, etc.) or a protein into a cell by various methods including but not limited to Agrobacterium transformation, gene gun bombardment, electroporation, PEG transformation, etc., so that the nucleic acid or protein can function in the cell.

[0094] In the present application, "ADC enzyme" refers to L-aspartate-α-decarboxylase.

[0095] In the present application, "ADC enzyme mutant" refers to an L-aspartate-α-decarboxylase mutant.

[0096] In the present application, "wild-type ADC enzyme" and "unmutated ADC enzyme" both refer to L-aspartate-α-decarboxylase having an amino acid sequence as shown in SEQ ID No.1.

[0097] In some embodiments, a L-aspartate-α-decarboxylase mutant is provided, wherein the L-aspartate-α-decarboxylase mutant comprises amino acid mutations at one or more sites compared with the L-aspartate-α-decarboxylase shown in SEQ ID No. 1.

[0098] The amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No.1 is derived from insects, and the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No.1 has been disclosed in the prior art CN115261364 A. In the prior art CN115261364 A, the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No.1 involved in the present invention is named CtADCN3.

[0099] In some embodiments, a L-aspartate-α-decarboxylase mutant is provided, which comprises an amino acid mutation at one or more of the following sites compared to the amino acid sequence of the L-aspartate-α-decarboxylase shown in SEQ ID No. 1:

[0100] No. 65, No. 70, No. 71, No. 87, No. 90, No. 110, No. 124, No. 128, No. 148, No. 203, No. 204, No. 209, No. 228, No. 229, No. 254, No. 284, No. 286, No. 291, No. 294, No. 347, No. 371, No. 392 and No. 463.

[0101] In some embodiments, the provided L-aspartate-α-decarboxylase mutant comprises an amino acid mutation at one or more of the following sites compared to the amino acid sequence of the L-aspartate-α-decarboxylase shown in SEQ ID No. 1:

[0102] No. 65, No. 70, No. 203, No. 204, No. 209, No. 254, No. 286, No. 291, No. 294, No. 347, No. 371, No. 392 and No. 463.

[0103] In some embodiments, the provided L-aspartate-α-decarboxylase mutant comprises any one of the following mutations or any suitable multiple mutations compared to the amino acid sequence shown in SEQ ID NO.1:

[0104] (1) Glycine 65 was mutated to asparagine (G65N),

[0105] (2) Glycine 65 mutated to serine (G65S),

[0106] (3) Alanine at position 70 mutated to aspartic acid (A70D),

[0107] (4) Alanine 70 mutated to glutamic acid (A70E),

[0108] (5) Alanine at position 70 mutated to glycine (A70G),

[0109] (6) Alanine 70 mutated to serine (A70S),

[0110] (7) Threonine at position 71 mutated to valine (T71V),

[0111] (8) Threonine 71 mutated to phenylalanine (T71F),

[0112] (9) Threonine 71 mutated to asparagine (T71N),

[0113] (10) Threonine 71 mutated to arginine (T71R),

[0114] (11) Lysine 87 mutated to histidine (K87H),

[0115] (12) Isoleucine at position 90 mutated to phenylalanine (I90F),

[0116] (13) Alanine at position 110 mutated to glutamine (A110Q),

[0117] (14) Methionine at position 124 mutated to leucine (M124L),

[0118] (15) Phenylalanine at position 128 mutated to threonine (F128T),

[0119] (16) Phenylalanine at position 128 mutated to alanine (F128A),

[0120] (17) Serine 148 mutated to alanine (S148A),

[0121] (18) Serine 148 mutated to glycine (S148G),

[0122] (19) Histidine at position 203 was mutated to glutamine (H203Q),

[0123] (20) Methionine at position 204 was mutated to asparagine (M204N),

[0124] (21) Methionine at position 204 mutated to arginine (M204R),

[0125] (22) Methionine at position 204 was mutated to valine (M204V),

[0126] (23) Threonine at position 209 mutated to serine (T209S),

[0127] (24) Threonine 209 mutated to tyrosine (T209Y),

[0128] (25) Threonine at position 209 mutated to glutamic acid (T209E),

[0129] (26) Tyrosine at position 228 mutated to valine (Y228V),

[0130] (27) Serine 229 mutated to alanine (S229A),

[0131] (28) Lysine 254 was mutated to methionine (K254M).

[0132] (29) Lysine 254 was mutated to glycine (K254G),

[0133] (30) Threonine at position 284 was mutated to alanine (T284A),

[0134] (31) Threonine at position 284 mutated to phenylalanine (T284F),

[0135] (32) Threonine at position 284 mutated to tyrosine (T284Y),

[0136] (33) Isoleucine at position 286 mutated to glutamine (I286Q),

[0137] (34) Proline 291 mutated to arginine (P291R),

[0138] (35) Glutamine at position 294 mutated to serine (Q294S),

[0139] (36) Glutamine at position 347 mutated to isoleucine (Q347I),

[0140] (37) Glutamine at position 347 mutated to leucine (Q347L),

[0141] (38) Glutamine at position 347 mutated to valine (Q347V),

[0142] (39) Leucine at position 371 mutated to valine (L371V),

[0143] (40) Leucine at position 371 mutated to isoleucine (L371I),

[0144] (41) Leucine 371 mutated to phenylalanine (L371F),

[0145] (42) Leucine 371 mutated to tyrosine (L371Y),

[0146] (43) Leucine at position 371 was mutated to aspartic acid (L371D),

[0147] (44) Glycine 392 mutated to isoleucine (G392I),

[0148] (45) Glycine at position 392 was mutated to valine (G392V),

[0149] (46) mutation of glycine 392 to phenylalanine (G392F), and

[0150] (47) Aspartic acid at position 463 was mutated to valine (D463V).

[0151] In some embodiments, a nucleic acid molecule is provided, which encodes the aforementioned L-aspartate-α-decarboxylase mutant.

[0152] In some embodiments, a recombinant expression vector is provided, comprising a nucleic acid molecule.

[0153] In some embodiments, a recombinant expression vector is provided that includes a plasmid.

[0154] In some embodiments, a vector cell is provided, comprising one or more of a nucleic acid molecule or a recombinant expression vector.

[0155] In some embodiments, provided vector cells include E. coli cells.

[0156] In some embodiments, a recombinant genetically engineered bacterium is provided, wherein the host cell used by the genetically engineered bacterium is Escherichia coli, preferably E. coli BL21 (DE3), but not limited to the E. coli host.

[0157] In some embodiments, the cDNA sequence of the L-aspartate-α-decarboxylase mutant is cloned into a separate expression vector and transformed into Escherichia coli to enhance the expression of the L-aspartate-α-decarboxylase mutant after adding an inducer.

[0158] In some embodiments, a method for producing an L-aspartate-α-decarboxylase mutant is provided, comprising the following steps: culturing the vector cell; and isolating the L-aspartate-α-decarboxylase mutant from the obtained culture.

[0159] In some embodiments, use of an L-aspartate-α-decarboxylase mutant or a vector cell in the production of β-alanine is provided.

[0160] In some embodiments, a method for preparing β-alanine is provided, comprising the following steps: using a culture of an L-aspartate-α-decarboxylase mutant or a carrier cell to catalyze a substrate to prepare β-alanine.

[0161] In a non-limiting manner, the gene encoding the L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is modified by site-directed mutagenesis, and the enzymatic activity of the L-aspartate-α-decarboxylase encoded thereby is increased, so that the catalytic performance of the genetically engineered bacteria expressing the ADC mutant is significantly improved. Highly active ADC enzymes can significantly reduce the amount of enzyme used, improve reaction efficiency, and thus reduce production costs, and have higher industrial application value.

[0162] The provided L-aspartate-α-decarboxylase mutant has high enzyme activity. When used for catalytic production of β-alanine, the substrate conversion rate can reach 99.9%, and there is almost no substrate residue in the system, which is convenient for industrial production.

[0163] The molecular biology experiments in the examples include preparation of host competent cells, construction of plasmids, restriction digestion and ligation of genes, plasmid transformation, PCR gene amplification and other experimental techniques, which are mainly carried out with reference to the Molecular Cloning Experiment Guide (3rd edition).

[0164] Example 1. Obtaining L-aspartate-α-decarboxylase mutants

[0165] A site-directed monosaturated mutation library was designed based on the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1, that is, the selected amino acid sites in the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 were mutated into 18 other amino acids in addition to the original amino acids and cysteine.

[0166] The plasmid in which the mutated gene was connected to the expression vector pET28a was obtained by the whole plasmid PCR method. The plasmid was digested with DpnI for about 3 hours and transferred into Escherichia coli E. coli DH5α using competent heat stimulation. Single clone colonies were selected and incubated overnight at 37°C and 200 rpm. The plasmid was extracted and sequenced, and the plasmid with the correct sequencing result was transferred into E. coli BL21 (DE3).

[0167] The successfully constructed mutant bacteria were screened for those with higher enzyme activity by high-throughput fluorescence method, and positive clones with improved partial activity compared with the unmutated ADC enzyme were initially obtained. The mutant genotype was determined by sequencing, and the amino acid sequence of the L-aspartate-α-decarboxylase mutant was determined. The obtained L-aspartate-α-decarboxylase mutant is shown in Table 1.

[0168] The operation method of measuring the enzyme activity of L-aspartate-α-decarboxylase mutant using high-throughput fluorescence method is as follows:

[0169] The 5ul glycerol tube was inoculated into 0.5ml medium in a 96-deep-well plate and cultured at 37°C and 800rpm for 18h. The 15% (v / v) inoculation volume was inoculated into 0.5ml 96-deep-well plate containing inducer medium and cultured at 30°C and 800rpm for 18h. The bacteria were collected by centrifugation, and 0.2ml Triton aqueous solution (2%, w / v) was added to each well of the 96-deep-well plate, cultured at 25°C and 800rpm for 1h, and then centrifuged at 10000g for 10min.

[0170] Place 50 µl of sodium L-aspartate solution (50 g / l) in a 96-well plate, then add 50 µl of the supernatant from the above-mentioned cell treatments, and incubate for 37 min. o C. Culture at 300 rpm for 0.5 h.

[0171] Add 155µl borate buffer (0.2M, pH8.5), 5µl o-diphenylmethanol solution, 5µl BME ethanol solution, and 5µl of the above enzyme reaction solution to a 96-well plate. Use the fluorescence mode of the microplate reader for kinetic detection, set the excitation wavelength (ex) to 360nm, the emission wavelength (em) to 460nm, and the gain value to 50. Detect once every 5 minutes and run for 4 hours. The change in fluorescence intensity over time is compared with the unmutated ADC enzyme to determine the enzyme activity. The relative activity of the mutant is calculated by the following method: the relative fluorescence intensity of the unmutated ADC enzyme WT is recorded as I0, the fluorescence intensity of the mutant is recorded as I, and the relative activity of the unmutated ADC enzyme WT is taken as 100. .

[0172] The relative enzyme activity test results of the obtained L-aspartate-α-decarboxylase mutants can be found in Table 1.

[0173] Table 1

[0174]

[0175] The relative activity of the unmutated ADC enzyme (L-aspartate-α-decarboxylase with an amino acid sequence as shown in SEQ ID No. 1) is 100, and the relative activity value of the L-aspartate-α-decarboxylase mutant enzyme activity indicates the degree of improvement. As shown in Table 1, the enzyme activity of the provided L-aspartate-α-decarboxylase mutant is improved by 19%-75%.

[0176] The amino acid sequence of SEQ ID No.1 is as follows:

[0177] .

[0178] In Table 1:

[0179] The mutant MJADC-001 is a mutant in which the 65th G of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to N;

[0180] The mutant MJADC-002 is a mutant in which the 65th G of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to S;

[0181] The mutant MJADC-003 is a mutant in which the 70th position A of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to D;

[0182] The mutant MJADC-004 is a mutant in which the 70th position A of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to E;

[0183] The mutant MJADC-005 is a mutant in which the 70th A in the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to G;

[0184] The mutant MJADC-006 is a mutant in which the 70th A in the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to S;

[0185] The mutant MJADC-007 is a mutant in which the 71st T of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to V;

[0186] The mutant MJADC-008 is a mutant in which the 71st T of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to F;

[0187] The mutant MJADC-009 is a mutant in which the 71st T of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to N;

[0188] The mutant MJADC-010 is a mutant in which the 71st T of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to R;

[0189] The mutant MJADC-011 is a mutant in which the 87th K of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to H;

[0190] The mutant MJADC-012 is a mutant in which the 90th I of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to F;

[0191] The mutant MJADC-013 is a mutant in which the 110th A in the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to Q;

[0192] The mutant MJADC-014 is a mutant in which the 124th M of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to L;

[0193] The mutant MJADC-015 is a mutant in which the 128th F of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to T;

[0194] The mutant MJADC-016 is a mutant in which the 128th F of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to A;

[0195] The mutant MJADC-017 is a mutant in which the 148th position S of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to A;

[0196] The mutant MJADC-018 is a mutant in which the 148th position S of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to G;

[0197] The mutant MJADC-019 is a mutant in which the 203rd position H of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to Q;

[0198] The mutant MJADC-020 is a mutant in which the 204th position M of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to N;

[0199] The mutant MJADC-021 is a mutant in which the 204th position M of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to R;

[0200] The mutant MJADC-022 is a mutant in which the 204th M of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to V;

[0201] The mutant MJADC-023 is a mutant in which the 209th T in the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to S;

[0202] The mutant MJADC-024 is a mutant in which the 209th T in the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to Y;

[0203] The mutant MJADC-025 is a mutant in which the 209th T of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to E;

[0204] The mutant MJADC-026 is a mutant in which the Y at position 228 of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to V;

[0205] The mutant MJADC-027 is a mutant in which the 229th position S of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to A;

[0206] The mutant MJADC-028 is a mutant in which the 254th K of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to M;

[0207] The mutant MJADC-029 is a mutant in which the 254th K of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to G;

[0208] The mutant MJADC-030 is a mutant in which the 284th T of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to A;

[0209] The mutant MJADC-031 is a mutant in which the 284th T of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to F;

[0210] The mutant MJADC-032 is a mutant in which the 284th T of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to Y;

[0211] The mutant MJADC-033 is a mutant in which the 286th I of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to Q;

[0212] The mutant MJADC-034 is a mutant in which the 291st P of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to R;

[0213] The mutant MJADC-035 is a mutant in which the 294th Q in the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to S;

[0214] The mutant MJADC-036 is a mutant in which the Q at position 347 of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to I;

[0215] The mutant MJADC-037 is a mutant in which the Q at position 347 of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to L;

[0216] The mutant MJADC-038 is a mutant in which the Q at position 347 of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to V;

[0217] The mutant MJADC-039 is a mutant in which the 371st L of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to V;

[0218] The mutant MJADC-040 is a mutant in which the 371st L of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to I;

[0219] The mutant MJADC-041 is a mutant in which the 371st L of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to F;

[0220] The mutant MJADC-042 is a mutant in which the 371st L of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to Y;

[0221] The mutant MJADC-043 is a mutant in which the 371st L of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to D;

[0222] The mutant MJADC-044 is a mutant in which the 392nd G of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to I;

[0223] The mutant MJADC-045 is a mutant in which the G at position 392 of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to V;

[0224] The mutant MJADC-046 is a mutant in which the 392nd G of the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is mutated to F;

[0225] The mutant MJADC-047 is obtained by mutating the 463rd D in the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 to V.

[0226] Example 2: Catalytic performance test of L-aspartate-α-decarboxylase mutant

[0227] The variant enzymes with different improved activities in Example 1 were selected, and the strains of the unmutated ADC enzyme and the mutant ADC enzyme were fermented and cultured and centrifuged, and the bacteria were added to 0.4 L of reaction solution at an addition amount of 12% (w / w) (ratio to the amount of substrate L-aspartic acid), the reaction solution substrate L-aspartic acid concentration was 200 g / L, pyridoxal phosphate (PLP) was added at a final concentration of 0.02 g / L, and the reaction was catalyzed at 37° C. and 300 rpm. A 2M sulfuric acid aqueous solution was added to the reaction solution to maintain pH 6.5, and samples were taken at regular intervals. The amount of substrate and product β-alanine generated was analyzed by HPLC. The HPLC method is as follows:

[0228] The high performance liquid chromatograph was Waters 2695-PDA996, ​​the chromatographic column was Phenomenex Luna C18 (250 mm × 4.6 mm × 5 μm) or equivalent chromatographic column, and the HPLC conditions were as follows: mobile phase A: water + 0.1% (v / v) acetic acid + 0.1% (v / v) triethylamine; mobile phase B: acetonitrile; flow rate: 0.8 ml / minute; detection wavelength: 360 nm; column temperature: 30 °C; 2,4-dinitrofluorobenzene (AR) pre-column derivatization. The elution gradient can be found in Table 2.

[0229] Table 2

[0230]

[0231] , the results are shown in Table 3 below:

[0232] Table 3

[0233]

[0234] As can be seen from Table 3 above, the variant enzyme with higher activity improvement has a higher substrate conversion rate at the same time. Under sufficient time, some L-aspartate-α-decarboxylase mutants, such as MJADC-002, MJADC-023, MJADC-035 and MJADC-029, can almost completely convert the substrate into the product, with a conversion rate of 99.9%, and have higher industrial application potential.

[0235] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0236] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. An L-aspartate-α-decarboxylase mutant, characterized in that: Compared with the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1, the L-aspartate-α-decarboxylase mutant comprises amino acid mutations at one or more of the following sites: No. 65, No. 70, No. 71, No. 87, No. 90, No. 110, No. 124, No. 128, No. 148, No. 203, No. 204, No. 209, No. 228, No. 229, No. 254, No. 284, No. 286, No. 291, No. 294, No. 347, No. 371, No. 392 and No.

463.

2. The L-aspartate-α-decarboxylase mutant according to claim 1, characterized in that Including amino acid mutations at one or more of the following sites: No. 65, No. 70, No. 203, No. 204, No. 209, No. 254, No. 286, No. 291, No. 294, No. 347, No. 371, No. 392 and No.

463.

3. The L-aspartate-α-decarboxylase mutant according to claim 1, characterized in that The L-aspartate-α-decarboxylase mutant includes the following mutations or any suitable multiple mutations: (1) Glycine at position 65 was mutated to asparagine, (2) Glycine at position 65 mutated to serine, (3) Alanine at position 70 mutated to aspartic acid, (4) Alanine at position 70 mutated to glutamic acid, (5) Alanine at position 70 mutated to glycine, (6) Alanine at position 70 mutated to serine, (7) Threonine at position 71 mutated to valine, (8) Threonine at position 71 mutated to phenylalanine, (9) Threonine at position 71 mutated to asparagine, (10) Threonine at position 71 mutated to arginine, (11) Lysine at position 87 mutated to histidine, (12) Isoleucine at position 90 mutated to phenylalanine, (13) Alanine at position 110 mutated to glutamine, (14) Methionine at position 124 mutated to leucine, (15) Phenylalanine at position 128 mutated to threonine, (16) Phenylalanine at position 128 mutated to alanine, (17) The serine at position 148 was mutated to alanine, (18) The serine at position 148 was mutated to glycine, (19) The histidine at position 203 was mutated to glutamine, (20) Methionine at position 204 was mutated to asparagine, (21) Methionine at position 204 mutated to arginine, (22) Methionine at position 204 mutated to valine, (23) Threonine at position 209 mutated to serine, (24) Threonine at position 209 mutated to tyrosine, (25) Threonine at position 209 mutated to glutamic acid, (26) Tyrosine at position 228 mutated to valine, (27) The serine at position 229 was mutated to alanine, (28) Lysine 254 was mutated to methionine, (29) Lysine 254 was mutated to glycine, (30) Threonine at position 284 was mutated to alanine, (31) Threonine at position 284 mutated to phenylalanine, (32) Threonine at position 284 mutated to tyrosine, (33) Isoleucine at position 286 was mutated to glutamine. (34) Proline 291 mutated to arginine, (35) Glutamine at position 294 mutated to serine, (36) Glutamine at position 347 mutated to isoleucine, (37) Glutamine at position 347 mutated to leucine, (38) Glutamine at position 347 mutated to valine, (39) Leucine at position 371 mutated to valine, (40) Leucine at position 371 mutated to isoleucine, (41) Leucine at position 371 mutated to phenylalanine, (42) Leucine at position 371 mutated to tyrosine, (43) Leucine at position 371 mutated to aspartic acid, (44) Glycine at position 392 mutated to isoleucine, (45) Glycine at position 392 was mutated to valine, (46) Glycine 392 mutated to phenylalanine, and (47) Aspartic acid at position 463 was mutated to valine.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the L-aspartate-α-decarboxylase mutant according to any one of claims 1 to 3.

5. A recombinant expression vector, characterized in that: Comprising the nucleic acid molecule of claim 4; optionally, the recombinant expression vector comprises a plasmid.

6. A vector cell, characterized in that: Contains one or more of the nucleic acid molecule of claim 4 or the recombinant expression vector of claim 5.

7. The vector cell according to claim 6, characterized in that The vector cells include Escherichia coli cells.

8. A method for producing an L-aspartate-α-decarboxylase mutant, comprising the steps of: culturing the vector cell according to claim 6 or 7; and isolating the L-aspartate-α-decarboxylase mutant from the obtained culture.

9. Use of the L-aspartate-α-decarboxylase mutant according to any one of claims 1 to 3 or the vector cell according to claim 6 or 7 in the preparation of β-alanine.

10. A method for preparing β-alanine, characterized in that: The method comprises the following steps: using the L-aspartate-α-decarboxylase mutant according to any one of claims 1 to 3 or the culture of the carrier cell according to claim 6 or 7 to catalyze the substrate to prepare β-alanine.

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