Glutamate decarboxylase mutant as well as preparation method and application thereof

Abstract

The invention provides a glutamate decarboxylase mutant as well as a preparation method and application thereof, and relates to the technical field of biology. Compared with wild-type glutamate decarboxylase, the glutamate decarboxylase mutant provided by the invention has higher enzyme activity, has important significance on production of gamma-aminobutyric acid, and can obviously improve the production efficiency of gamma-aminobutyric acid.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a glutamate decarboxylase mutant, a preparation method thereof, and an application thereof. Background Art

[0002] γ-aminobutyric acid (GABA) is a four-carbon non-protein amino acid, which is obtained by irreversibly and specifically removing one molecule of CO2 from the α-carboxyl group of sodium L-glutamate by glutamate decarboxylase (GAD). Currently, it is widely used in the industrial, animal feed, pharmaceutical, and food industries.

[0003] The methods for obtaining GABA include chemical synthesis and biosynthesis. Chemical synthesis has violent reactions, low yields, many by-products, is difficult to purify, and the products lack safety, so it is not suitable as a food additive. Biosynthesis methods include plant enrichment and microbial fermentation. Among them, microbial fermentation has the characteristics of safety, low cost, fast speed, simple cultivation, wide distribution, etc. Therefore, biosynthesis to produce food or pharmaceutical grade GABA is an ideal way. In particular, using lactic acid bacteria and yeast to ferment and produce GABA has the characteristics of low cost, simple tools, and high efficiency, and is suitable for large-scale production. However, the enzyme activity of the current wild-type glutamate decarboxylase is not yet ideal, and developing an enzyme with higher catalytic activity is an effective way to further improve the production efficiency of γ-aminobutyric acid.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] The first object of the present invention is to provide a glutamate decarboxylase mutant to solve the above technical problems.

[0006] The second object of the present invention is to provide a nucleic acid molecule.

[0007] The third object of the present invention is to provide an expression cassette.

[0008] The fourth object of the present invention is to provide a recombinant vector.

[0009] The fifth object of the present invention is to provide a cell.

[0010] The sixth object of the present invention is to provide a preparation method of the above glutamate decarboxylase mutant.

[0011] The seventh object of the present invention is to provide an application of the above glutamate decarboxylase mutant in the preparation of γ-aminobutyric acid.

[0012] In order to achieve the above objects, the following technical solutions are specifically adopted:

[0013] In a first aspect, the present invention provides a glutamate decarboxylase mutant, which is any one selected from the following mutants:

[0014] Mutant 1, the amino acid sequence of which is shown in SEQ ID NO.1;

[0015] Mutant 2, the amino acid sequence of which is shown in SEQ ID NO.2;

[0016] Mutant 3, the amino acid sequence of which is shown in SEQ ID NO.3.

[0017] In a second aspect, the present invention provides a nucleic acid molecule, which comprises a nucleotide sequence encoding the glutamate decarboxylase mutant.

[0018] As a further technical solution, the nucleotide sequence encoding Mutant 1 is shown in SEQ ID NO.4;

[0019] The nucleotide sequence encoding Mutant 2 is shown in SEQ ID NO.5;

[0020] The nucleotide sequence encoding Mutant 3 is shown in SEQ ID NO.6.

[0021] In a third aspect, the present invention provides an expression cassette, which contains the nucleic acid molecule.

[0022] In a fourth aspect, the present invention provides a recombinant vector, which contains the expression cassette.

[0023] As a further technical solution, the copy number of the expression cassette is 1-5.

[0024] In a fifth aspect, the present invention provides a cell, which carries the nucleic acid molecule, or contains the expression cassette, or contains the recombinant vector, or expresses the glutamate decarboxylase mutant.

[0025] As a further technical solution, the cell is Pichia pastoris GS115.

[0026] In a sixth aspect, the present invention provides a method for preparing the glutamate decarboxylase mutant, comprising: fermenting the cell and preparing the glutamate decarboxylase mutant after separation.

[0027] In a seventh aspect, the present invention provides the use of the glutamate decarboxylase mutant in the preparation of γ-aminobutyric acid.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The glutamate decarboxylase mutant provided by the present invention has higher enzyme activity compared with the wild-type glutamate decarboxylase, which is of great significance for the production of γ-aminobutyric acid and can significantly improve the production efficiency of γ-aminobutyric acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0031] Figure 1 is a vector construction map;

[0032] Figure 2 is a schematic diagram of multi-copy expression cassette construction;

[0033] Figure 3 is a DNA double digestion verification map; M: Maker; 1: single-copy target gene; 2: two-copy target gene; 3: three-copy target gene; 4: four-copy target gene; 5: five-copy target gene;

[0034] Figure 4 is an SDS-PAGE map for analyzing protein expression levels; M: Maker; 1: negative control without the target gene; 2: single-copy protein expression level; 3: two-copy protein expression level; 4: three-copy protein expression level; 5: four-copy protein expression level; 6: five-copy protein expression level;

[0035] Figure 5 is the reaction principle diagram of the present invention;

[0036] Figure 6 is the high performance liquid chromatography (HPLC) map after the reaction;

[0037] Figure 7 is the standard curve of sodium L-glutamate;

[0038] Figure 8 is the standard curve of γ-aminobutyric acid;

[0039] Figure 9 is the production rate map of multi-copy glutamate decarboxylase Pichia pastoris whole cells. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0040] The embodiments of the present invention will be described in detail below in combination with embodiments and examples. However, those skilled in the art will understand that the following embodiments and examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention. Those not specified in specific conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments not specified in the manufacturer are all conventional products that can be obtained through commercial purchase.

[0041] In a first aspect, the present invention provides a glutamate decarboxylase mutant, which is any one selected from the following mutants:

[0042] Mutant 1, the amino acid sequence is as shown in SEQ ID NO.1:

[0043] MGDKKQVTDLRSELLDSRFGAKSISTIAESKRFPLHEMRDDVAFQIINDELYLDGNARQNLATFCQTWDDENVHKLMDLSINKNWIAKEPYPQSAAIDLRCVNMVADLWHAPAPKNGQAVGTNTIGSSEACMLGGMAMKWRWRKRMEAAGKPTDKPNLVCGPVQICWHKFARYWDVELREIPMRPGQLFMDPKRMIEACDENTIGVVPTFGVTYTGNYEFPQPLHDALDKFQADTGIDIDMHIDAASGGFLAPFVAPDIVWDFRLPRVKSISASGHKFGLAPLGCGWVIWRDEEALPQELVFNVDYLGGRIGTFAINFSRPAGQVIAQYYEFLRLGREGYTKVQNASYQVAAYLADEIAKLGPYEFICTGRPDEGIPAVCFKLKDGEDPGYTLYDLSERLRLRGWQVPAFTLGGEATDIVVMRIMCRRGFEMDFAELLLEDYKASLKYLSDHPKLQGIAQQNSFKHT(SEQ ID NO.1).

[0044] Mutant 2, the amino acid sequence is as shown in SEQ ID NO.2:

[0045] MGDKKQVTDLRSELLDSRFGAKSISTIAESKRFPLHEMRDDVAFQIINDELYLDGNARQNLATFCQTWDDENVHKLMDLSINKNWINKEMYPQSAAIDLRCVNMVADLWHAPAPKNGQAVGTNTIGSSEACMLGGMAMKWRWRKRMEAAGKPTDKPNLVCGPVQICWHKFARYWDVELREIPMRPGQLFMDPKRMIEACDENTIGVVPTFGVTYTGNYEFPQPLHDALDKFQADTGIDIDMHIDAASGGFLAPFVAPDIVWDFRLPRVKSISASGHKFGLAPLGCGWVIWRDEEALPQELVFNVDYLGGHIGTFAINFSRPAGQVIAQYYEFLRLGREGYTKVQNASYQVAAYLADEIAKLGPYEFICTGRPDEGIPAVCFKLKDGEDPGYTLYDLSERLRLRGWQVPAFTLGGEATDIVVMRIMCRRGFEMDFAELLLEDYKASLKYLSDHPKLQGIAQQNSFKHT(SEQ ID NO.2).

[0046] Mutant 3, the amino acid sequence is shown in SEQ ID NO.3:

[0047] MGDKKQVTDLRSELLDSRFGAKSISTIAESKRFPLHEMRDDVAFQIINDELYLDGNARQNLATFCQTWDDENVHKLMDLSINKNWIAKEPYPQSAAIDLRCVNMVADLWHAPAPKNGQAVGTNTIGSSEACMLGGMAMKWRWRKRMEAAGKPTDKPNLVCGPVQICWHKFARYWDVELREIPMRPGQLFMDPKRMIEACDENTIGVVPTFGVTYTGNYEFPQPLHDALDKFQADTGIDIDMHIDAASGGFLAPFVAPDIVWDFRLPRVKSISASGHKFGLAPLGCGWVIWRDEEALPQELVFNVDYLGGAIGTFAINFSRPAGQVIAQYYEFLRLGREGYTKVQNASYQVAAYLADEIAKLGPYEFICTGRPDEGIPAVCFKLKDGEDPGYTLYDLSERLRLRGWQVPAFTLGGEATDIVVMRIMCRRGFEMDFAELLLEDYKASLKYLSDHPKLQGIAQQNSFKHT(SEQ ID NO.3).

[0048] It has been found by the inventors that the glutamate decarboxylase mutant provided by the present invention has higher enzyme activity compared to the wild-type glutamate decarboxylase and can be used for the production of γ-aminobutyric acid.

[0049] In a second aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the glutamate decarboxylase mutant.

[0050] This nucleic acid molecule can be translated to obtain the glutamate decarboxylase mutant of the present invention.

[0051] In some alternative embodiments, the nucleotide sequence encoding Mutant 1 is as shown in SEQ ID NO.4:

[0052]

[0053] The nucleotide sequence encoding the mutant 2 is shown in SEQ ID NO. 5:

[0054]

[0055] The nucleotide sequence encoding the mutant 3 is shown in SEQ ID NO.6:

[0056]

[0057] In the present invention, the sequence of the nucleic acid molecule encoding the glutamate decarboxylase mutant has been codon-optimized, which can significantly improve the expression efficiency of the glutamate decarboxylase mutant.

[0058] In a third aspect, the present invention provides an expression cassette, which contains the nucleic acid molecule described above.

[0059] This expression cassette can be translated to obtain the glutamate decarboxylase mutant of the present invention.

[0060] In a fourth aspect, the present invention provides a recombinant vector, which contains the expression cassette described above.

[0061] By introducing this vector into a recipient cell, the recipient cell can express the glutamate decarboxylase mutant of the present invention.

[0062] In some alternative embodiments, the vector includes but is not limited to plasmids.

[0063] In some alternative embodiments, the copy number of the expression cassette is 1 - 5. Through research by the inventors, it has been found that when the copy number of the expression cassette is 4, after being introduced into the recipient cell, the expression level of the glutamate decarboxylase mutant is the highest.

[0064] In a fifth aspect, the present invention provides a cell, which carries the nucleic acid molecule described above, or contains the expression cassette described above, or contains the recombinant vector described above, or expresses the glutamate decarboxylase mutant described above.

[0065] In some alternative embodiments, the cell is Pichia pastoris GS115.

[0066] In a sixth aspect, the present invention provides a method for preparing the glutamate decarboxylase mutant described above, including: fermenting the cell described above, and preparing the glutamate decarboxylase mutant after separation.

[0067] This preparation method is simple and efficient, and a large amount of glutamate decarboxylase mutant can be obtained through fermentation.

[0068] In a seventh aspect, the present invention provides the application of the glutamate decarboxylase mutant described above in the preparation of γ-aminobutyric acid.

[0069] The glutamate decarboxylase mutant provided by the present invention has higher enzyme activity compared with the wild-type glutamate decarboxylase, and can be used for the production of γ-aminobutyric acid.

[0070] The present invention will be further illustrated by specific examples below. However, it should be understood that these examples are only for more detailed illustration and should not be construed as limiting the present invention in any form.

[0071] Example 1: Construction of a single-copy expression vector for glutamate decarboxylase

[0072] The vector construction map is as Figure 1 shown.

[0073] Step 1: Amplify the fragment using the plasmid pRS426-gadB containing the target gene sequence (glutamate decarboxylase gene from Escherichia coli, GenBank number at NCBI is NC_000913.3) stored in the laboratory as a template. The upstream primer sequence R used is: 5′-TTATTCGAAACGAGGAATTCGCCACCATGGGTGATAAGAAGCAAGT CACT-3′ (SEQ ID NO.7), and the downstream primer sequence R is: 5′-TGGCATTCTGACATCCTC TTGATCAAGTGTGCTTGAAAGAGTTTTGTTG-3′ (SEQ IDNO.8). The PCR reaction system (50 μL): 1 μL of plasmid; 1 μL of PrimeSTAR GXL DNA Polymerase; 1.5 μL of upstream primer F; 1.5 μL of downstream primer R; 4 μL of dNTPs; 10 μL of 5×PCR Buffer; the remaining volume is made up with ddH2O. The PCR reaction program: pre-denaturation at 94°C for 2 min; denaturation at 95°C for 10 s, annealing at 58°C for 30 s, fragment extension at 68°C for 1.5 min, and the number of cycles is 30 times.

[0074] PCR amplify the vector using pAO815 as a template. The upstream primer sequence F is: 5′-CAAAACTCTT TCAAGCACACTTGATCAAGAGGATGTCAGAATGCCATT-3′ (SEQ IDNO.9), and the vector downstream primer sequence R is: 5′-TCAGTGACTTGC TTCTTATCACCCATGGTGGCGAATTCCTCGTTTCGAAT-3′ (SEQ ID NO.10). The PCR reaction system (50 μL): 1 μL of plasmid; 1 μL of PrimeSTAR GXL DNA Polymerase; 1.5 μL of upstream primer F; 1.5 μL of downstream primer R; 4 μL of dNTPs; 10 μL of 5×PCR Buffer; the remaining volume is made up with ddH2O. The PCR reaction program: pre-denaturation at 94°C for 2 min; denaturation at 95°C for 10 s, annealing at 58°C for 30 s, vector extension at 68°C for 7.5 min, and the number of cycles is 30 times.

[0075] Step 2: Purify and recover the fragments and vectors according to the operation instructions of the Tiangen Universal DNA Purification and Recovery Kit. After ligating the purified fragments and vectors by homologous recombination, transform them into DH5α competent cells and spread them on an LB plate containing the corresponding Amp antibiotic, and culture overnight at 37°C. Pick a single colony into 10 mL of LB liquid medium, shake and culture for 7 - 9 h, and take an appropriate amount of the bacterial liquid for sequencing. After successful sequencing, extract the plasmid to obtain a single-copy expression vector of glutamate decarboxylase.

[0076] Example 2: Construction of Glutamate Decarboxylase Mutants

[0077] Step 1: Design site-directed mutagenesis primers according to the whole plasmid amplification method, with the primer direction all being 5’—3’, and the primers are listed in Table 1.

[0078] Table 1 Mutagenesis Primers

[0079] F-87A / 90P AAGAACTGGATTgctAAGGAAccaTACCCAC(SEQ ID NO.11) R-87A / 90P TGTGGGTAtggTTCCTTagcAATCCAGTTC(SEQ ID NO.12) F-87N / 90M AACAAGAACTGGATTaacAAGGAAatgTACCC(SEQ ID NO.13) R-87N / 90M TGGGTAcatTTCCTTgttAATCCAGTTCTTG(SEQ ID NO.14) F-310R GACTACTTGGGTGGTagaATTGGTACCTTTGC(SEQ ID NO.15) R-310R TGCAAAGGTACCAATtctACCACCCAAGTAG(SEQ ID NO.16) F-310H GACTACTTGGGTGGTcatATTGGTACCTTTGC(SEQ ID NO.17) R-310H TGCAAAGGTACCAATatgACCACCCAAGTAG(SEQ ID NO.18) F-310A GACTACTTGGGTGGTgctATTGGTACCTTTGC(SEQ ID NO.19) R-310A TGCAAAGGTACCAATagcACCACCCAAGTAG(SEQ ID NO.20)

[0080] Step 2: Obtain mutant plasmids according to the PCR reaction system in Table 2. Subsequently, add 1 μL of DpnⅠ to the PCR product to eliminate the template plasmid. After purification, take 3 μg of the linearized plasmid and integrate it into Pichia pastoris GS115. Spread the obtained recombinant Pichia pastoris on an MD plate and culture at 30°C for 3 days. Then pick monoclonal strains for sequencing, and induce and express the correctly sequenced monoclonal strains to measure the enzyme activity. The effects of amino acid mutations on the activity are shown in Table 3. A total of 3 mutants were obtained, named Mutant 1 (nucleotide sequence as shown in SEQ ID NO.4), Mutant 2 (nucleotide sequence as shown in SEQ ID NO.5), and Mutant 3 (nucleotide sequence as shown in SEQ ID NO.6). As can be seen from Table 3, Mutant 3 of the protein has the highest enzyme activity. Therefore, this mutant gene is used for multi-copy expression in the subsequent experimental process.

[0081] Table 2 PCR Reaction System

[0082]

[0083] Table 3 Effects of Amino Acid Mutations on Enzyme Activity

[0084] Strain type Mutation position Enzyme activity (U / mL) Multiple of wild type Wild type —— 94.5 1 Mutant 1 D87A / E90P / Q310R 138.7 1.47 Mutant 2 D87N / E90M / Q310H 146.5 1.55 Mutant 3 D87A / E90P / Q310A 152.1 1.61

[0085] In this example, the method for measuring enzyme activity: At 50°C, in a 500 μL reaction system, add 10 μL of enzyme solution (the mutant protein concentration in each enzyme solution is the same), and the remaining 490 μL is the reaction solution. The reaction solution is a sodium acetate buffer containing 50 μM substrate L-sodium glutamate and 0.8 μM coenzyme PLP, with a pH of 5. The GABA content is measured by HPLC.

[0086] Enzyme activity definition (U): The amount of enzyme required to produce 1 μmol of GABA per minute under specific conditions.

[0087] Example 3: Construction of a multi-copy expression vector for glutamate decarboxylase

[0088] Schematic diagram of the construction of the multi-copy expression cassette is as Figure 2 shown.

[0089] Step 1: Double-digest the single-copy expression vector of glutamate decarboxylase with BglII and BamhI to obtain the glutamate decarboxylase gene expression cassette fragment. Single-digest the single-copy expression vector of glutamate decarboxylase with BamhI, and recover and purify the vector fragment by gel electrophoresis. Utilizing the property that BglII and BamhI are isocaudomers, ligate the vector fragment and the glutamate decarboxylase gene expression cassette fragment with T4 ligase. The process of constructing a 2-copy glutamate decarboxylase expression vector by the isocaudomer method is as Figure 2 shown. Transform it into DH5α competent cells by the chemical method. Spread it on an LB plate containing Amp and culture it overnight at 37 °C. Pick a single colony into 5 mL of LB liquid medium, shake and culture for 7 - 9 h, extract the plasmid, and double-digest the plasmid to verify whether the plasmid is successfully constructed. Name the expression vector containing 2 copies of the glutamate decarboxylase gene expression cassette as GS115-pAO815-gadB-2ca.

[0090] Step 2: Double-digest and purify the 2-copy vector with BglII and BamhI to obtain the glutamate decarboxylase 2-copy gene expression cassette. Connect and culture the vector from Step 1 and the 2-copy gene expression cassette according to Step 1. Finally, obtain an expression vector containing 3 copies of the glutamate decarboxylase gene expression cassette, named GS115-pAO815-gadB-3ca, and construct GS115-pAO815-gadB-4ca and GS115-pAO815-gadB-5ca in the same way. Subsequently, perform double-digestion verification with BglII and BamhI, and the results are as Figure 3 shown.

[0091] Example 4: Construction and expression of Pichia pastoris with 1 - 5 copies of glutamate decarboxylase

[0092] Step 1: Linearize the constructed single-copy and multi-copy expression vectors using StuI. The enzyme digestion reaction system is 50 μL, including 5 - 10 μg of recombinant plasmid, 2 μL of StuI restriction endonuclease, 5 μL of 10× buffer, and ddH2O to make up the remaining volume. Incubate in a 37°C water bath for 30 minutes. Immediately after the reaction, perform agarose gel electrophoresis and recover the linearized product. Then, mix 3 - 5 μg of linearized plasmid DNA with competent cells, transfer to an electroporation cuvette, and incubate on ice for 5 minutes. Set the electroporation parameters to 2 kV, 25 μF, and 200 Ω. Immediately after electroporation, add 1 mL of pre-cooled 1 M sorbitol solution, gently pipette to mix evenly, transfer to a 1.5 mL centrifuge tube, and incubate at 30°C for 1 hour. Then, add 1 mL of YPD medium and incubate with shaking at 30°C and 180 rpm for 1 hour. After incubation, centrifuge at 4000 rpm for 1 minute, discard part of the supernatant, take 100 μL of the evenly suspended bacterial solution and spread it on an MD solid medium, and incubate in a 30°C constant temperature incubator for 2 - 4 days, then pick positive transformants.

[0093] Step 2: Pick monoclonal colonies on the MD resistance plate and inoculate them into 5 mL of YPD medium, and incubate at 30°C and 180 rpm until the logarithmic growth phase. Then, transfer with an inoculation amount of 1% to 100 mL of BMGY medium and incubate for 24 h. Centrifuge at 4000 rpm for 3 min, discard the supernatant, and resuspend the cells with pre-cooled sterile water 2 - 3 times. Resuspend the cells in 100 mL of BMMY medium and induce incubation at 30°C and 180 rpm for 3 days. Add 1% methanol every 24 h for 3 days, then centrifuge to collect the bacteria. Resuspend the bacteria with sodium acetate buffer with a pH of 5, break the cells using glass beads, and then analyze the protein content in the suspension by SDS-PAGE. The results are as Figure 4 shown. The molecular weight of the secreted expression of Pichia pastoris with 1 - 5 copies of glutamate decarboxylase is approximately 52 kDa ( Figure 4 ). It was found that the protein expression level of Pichia pastoris with 4 copies of glutamate decarboxylase was the highest.

[0094] Example 5: Determination of the production rate of γ-aminobutyric acid from L-glutamate by whole-cell catalysis of glutamate decarboxylase

[0095] Step 1: Screen the above strains through the unit cell production rate, and select the strains that catalyze the production of γ-aminobutyric acid from L-glutamate (the reaction principle is as Figure 5The strain with the highest production rate (as shown) was defined, and the GABA production rate formula was: production rate = actual amount of GABA produced / theoretical amount of GABA produced. A total of 100 mL of the whole-cell catalysis system of glutamate decarboxylase was set up, containing 1 M sodium L-glutamate, 0.016 M coenzyme PLP, and 5 g / L of the cells. The reaction was carried out in a water bath at 50 °C and pH = 5 for 10 h, and samples were taken every 2 h to measure the production rate. Each reaction had 3 replicates. The product production rate was determined by HPLC. After the reaction ended, the product was detected by liquid chromatography. The results of the four-copy catalytic synthesis are as Figure 6 shown. It can be seen from the figure that with PLP as the coenzyme, sodium L-glutamate was successfully converted into γ-aminobutyric acid under the catalysis of glutamate decarboxylase.

[0096] Step 2: Solutions of different concentrations of the substrate sodium L-glutamate and the product γ-aminobutyric acid were prepared respectively. After HPLC detection, the standard curves of the two were obtained. The standard curves are as Figure 7 and Figure 8 shown, and the regression equations are Y = 267117*X + 35493, R 2 = 0.9959 and Y = 378804*X - 145840, R 2 = 0.9931, where x represents the product peak area and y represents the product concentration, with the unit of g / L.

[0097] Step 3: The catalytic results of different copies are shown in Table 4 and Figure 9 shown. It was found that the production rate and reaction rate of the whole cells of Pichia pastoris with four copies of glutamate decarboxylase were the highest.

[0098] Table 4 GABA production rate

[0099]

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A glutamate decarboxylase mutant, characterized in that, Any one selected from the following mutants: Mutant 1, with the amino acid sequence shown in SEQ ID NO.1; Mutant 2, with the amino acid sequence shown in SEQ ID NO.2; Mutant 3, with the amino acid sequence shown in SEQ ID NO.

3.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule comprises a nucleotide sequence encoding the glutamate decarboxylase mutant as claimed in claim 1.

3. The nucleic acid molecule according to claim 2, wherein, The nucleotide sequence encoding Mutant 1 is shown in SEQ ID NO.4; The nucleotide sequence encoding Mutant 2 is shown in SEQ ID NO.5; The nucleotide sequence encoding Mutant 3 is shown in SEQ ID NO.

6.

4. An expression cassette, characterized in that, The expression cassette contains the nucleic acid molecule as claimed in claim 2 or 3.

5. A recombinant vector, characterized in that, The recombinant vector contains the expression cassette as claimed in claim 4.

6. The recombinant vector according to claim 5, wherein The copy number of the expression cassette is 1 - 5.

7. A cell, characterized in that, The cell carries the nucleic acid molecule as claimed in claim 2 or 3, or contains the expression cassette as claimed in claim 4, or contains the recombinant vector as claimed in claim 5 or 6, or expresses the glutamate decarboxylase mutant as claimed in claim 1.

8. The cell according to claim 7, characterized in that, The cell is Pichia pastoris GS115.

9. The preparation method of the glutamate decarboxylase mutant according to claim 1, characterized in that, Comprising: Fermenting the cell as claimed in claim 7 or 8, and preparing the glutamate decarboxylase mutant after separation.

10. Use of the glutamate decarboxylase mutant as claimed in claim 1 in the preparation of γ-aminobutyric acid.

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