High-activity glutamate decarboxylase mutant and application thereof

Abstract

The invention provides a high-activity glutamic acid decarboxylase mutant and application thereof, the glutamic acid decarboxylase mutant is obtained by performing site-specific mutagenesis on wild-type glutamic acid decarboxylase derived from bacillus megatherium, and specifically, histidine at the 466th site is mutated into threonine. Compared with wild-type glutamate decarboxylase, the glutamic acid decarboxylase mutant constructed by the invention has the advantages that the enzyme activity is improved by 50.03%, the enzyme activity is improved by 4.1% compared with other glutamic acid decarboxylase mutants, the enzyme activity of the glutamic acid decarboxylase mutant constructed by the invention is the highest under the condition that the pH is 5-6, and after the glutamic acid decarboxylase mutant is incubated for 6 hours at the temperature of 50 DEG C, the enzyme activity of the glutamic acid decarboxylase mutant is greatly improved. The glutamic acid decarboxylase mutant constructed by the invention still keeps the catalytic activity of 45.9%. In conclusion, the glutamic acid decarboxylase mutant constructed by the invention effectively improves the enzyme activity and stability of glutamic acid decarboxylase, effectively solves the technical bottlenecks of insufficient catalytic efficiency, poor thermal stability and the like of an enzyme preparation in the existing production process, and has important application value for promoting the industrial production of GABA (gamma-aminobutyric acid).

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a highly active glutamate decarboxylase mutant and application thereof. Background Art

[0002] γ-Aminobutyric acid (GABA) is a naturally occurring, functional, non-protein amino acid with benefits such as enhancing brain function, lowering blood pressure, relieving anxiety, regulating endocrine function, controlling epileptic seizures, and protecting the liver and kidneys. It exhibits significant market potential in industries such as food, healthcare, beverage manufacturing, and beauty. Furthermore, GABA has garnered significant attention as a precursor for the synthesis of 2-pyrrolidone and the biodegradable material polyamide nylon 4, demonstrating its significant industrial applications.

[0003] Currently, GABA production processes primarily include chemical synthesis, microbial fermentation, and biotransformation. However, chemical synthesis suffers from high production costs and low yields. The resulting GABA contains residual raw materials, resulting in poor safety and unsuitable for use in sensitive areas such as food and pharmaceuticals. Microbial fermentation, while mild, safe, and low-cost, presents challenges such as long production cycles, suboptimal yields, and complex subsequent purification processes, hindering its industrialization. In contrast, biotransformation technology, with its advantages of simple operation, mild reaction conditions, high substrate utilization, excellent conversion efficiency, and low purification costs, is gradually becoming a focus of industry attention and is the primary direction for future GABA preparation process development.

[0004] Glutamate decarboxylase (GAD, EC4.1.1.15), a ubiquitous biocatalyst, efficiently and specifically catalyzes the irreversible conversion of L-glutamate (salt) into GABA. It is a core enzyme for biosynthesis of GABA and holds significant commercial development potential. However, existing research indicates that previously discovered glutamate decarboxylases generally suffer from low catalytic efficiency and an optimal pH that is slightly acidic, significantly limiting their potential for industrial application.

[0005] Addressing these technical bottlenecks, developing novel glutamate decarboxylases with superior performance and innovating GABA preparation processes will be key breakthroughs in achieving efficient GABA production. Improving the catalytic efficiency of glutamate decarboxylase through protein engineering techniques such as directed evolution combined with semi-rational design is of strategic importance for enhancing the enzyme's industrial application value. Summary of the Invention

[0006] Technical problems to be solved: In view of the above technical problems, the first object of the present invention is to provide a highly active glutamate decarboxylase mutant.

[0007] The second object of the present invention is to provide a nucleotide sequence of the glutamate decarboxylase mutant.

[0008] The third object of the present invention is to provide a recombinant expression vector containing the glutamate decarboxylase mutant. The fourth object of the present invention is to provide a recombinant cell containing the glutamate decarboxylase mutant or nucleotide sequence or recombinant expression vector.

[0009] The fifth object of the present invention is to provide the use of the glutamate decarboxylase mutant or nucleotide sequence or recombinant expression vector or recombinant cell in the biosynthesis of γ-aminobutyric acid.

[0010] Technical solution:

[0011] A high-activity glutamate decarboxylase mutant is obtained by mutating the histidine at position 466 of the wild-type glutamate decarboxylase derived from Bacillus megaterium to threonine.

[0012] Preferably, the amino acid sequence of the wild-type glutamate decarboxylase is shown in SEQ ID NO: 1.

[0013] Preferably, the nucleotide sequence encoding the glutamate decarboxylase mutant is shown in SEQ ID NO: 2.

[0014] A recombinant expression vector contains the glutamate decarboxylase mutant or the nucleotide sequence.

[0015] Preferably, the recombinant expression vector is a prokaryotic expression vector, including but not limited to pET21a and pXMJ19.

[0016] A recombinant cell contains the glutamate decarboxylase mutant or nucleotide sequence or recombinant expression vector.

[0017] Preferably, the recombinant cell is a prokaryotic cell, including but not limited to Escherichia coli and Corynebacterium glutamicum.

[0018] The use of the above-mentioned glutamate decarboxylase mutant or nucleotide sequence or recombinant expression vector or recombinant cell in the biosynthesis of γ-aminobutyric acid includes the steps of preparing the glutamate decarboxylase mutant or nucleotide sequence or recombinant expression vector or recombinant cell to produce γ-aminobutyric acid, and collecting the produced γ-aminobutyric acid.

[0019] Beneficial effects:

[0020] The invention mutates the histidine at position 466 of the wild-type glutamate decarboxylase derived from Bacillus megaterium into threonine to construct a glutamate decarboxylase mutant L100, thereby improving the enzymatic activity and stability of the glutamate decarboxylase at different pH and temperatures. The glutamate decarboxylase mutant L100 constructed by the present invention has an enzyme activity 50.03% higher than that of the wild-type glutamate decarboxylase and an enzyme activity 4.1% higher than that of another glutamate decarboxylase mutant G100 (the 467th histidine H is mutated to serine S). Under pH conditions of 5-6, the enzyme activity of the glutamate decarboxylase mutant L100 constructed by the present invention is the highest compared with the wild-type glutamate decarboxylase and the glutamate decarboxylase mutant G100. Under the optimum pH condition of 5.0, the enzyme activity of the glutamate decarboxylase mutant L100 constructed by the present invention is 29% higher than that of the wild-type and 9.3% higher than that of the glutamate decarboxylase mutant G100. After being kept warm at 50°C for 6 hours, the relative enzyme activity of the glutamate decarboxylase mutant L100 constructed by the present invention is 45.9%, which is 40.8% higher than that of the wild-type and 18.6% higher than that of the glutamate decarboxylase mutant G100. DETAILED DESCRIPTION

[0021] The present invention will be further described below in conjunction with examples, which are provided to explain the present invention and are not limited to the following examples:

[0022] Example 1

[0023] This example describes the construction of a glutamate decarboxylase mutant recombinant plasmid pET-21a-L100, comprising the following steps: synthesizing a glutamate decarboxylase mutant sequence L100 in which the histidine H at position 466 is mutated to the threonine T at GenScript Biotech Co., Ltd. based on the wild-type glutamate decarboxylase gene sequence of Bacillus megaterium, and inserting the L100 sequence into the pET-21a expression vector between the Bam HI and Hind III restriction sites to obtain the recombinant plasmid pET-21a-L100.

[0024] Example 2

[0025] This example is the construction of the glutamate decarboxylase mutant expression strain L100, comprising the following steps:

[0026] S1. The culture medium required for the transformation experiment is as follows: LB liquid medium: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L.

[0027] S2. 1 μL of the recombinant plasmid pET-21a-L100 constructed in Example 1 was added to 100 μL of competent E. coli BL21 cells, placed on ice for 40 min, heat-shocked at 42°C for 90 s, placed on ice for 4 min, added with 800 μL of LB liquid medium, incubated on a shaker at 37°C for 1 h, centrifuged, most of the supernatant was discarded, and about 100 μL of the culture medium was mixed with the precipitate, spread onto an Amp plate (Amp+LB), and incubated at 37°C for about 12 h to obtain a highly active mutant glutamate decarboxylase expressing strain L100;

[0028] S3. Pick a single colony and inoculate it into 5 mL LB liquid medium, culture for 12 h, inoculate 3-5% of the inoculum into 100 mL LB liquid medium (50 μg / mL Amp), and culture at 37°C, 180 rpm, shaking until the OD 600 =0.6, IPTG was added to a final concentration of 0.4 mmol / L, and the cells were induced at 25°C for 22 h. After the induction, the culture solution was centrifuged at 1700 rpm / min for 10 min to collect the cells.

[0029] Comparative Example 1

[0030] The difference between this comparative example and Example 2 is that this comparative example is the construction of the wild-type glutamate decarboxylase expression strain W100, and the specific process is the same as that of Examples 1-2.

[0031] Comparative Example 2

[0032] The difference between this comparative example and Example 2 is that this comparative example is the construction of the glutamate decarboxylase mutant expression strain G100, and the glutamate decarboxylase mutant G100 is a site-directed mutation of the histidine at position 467 of the wild-type glutamate decarboxylase to serine. The specific process is the same as that of Examples 1 and 2.

[0033] Index test:

[0034] 1. Determination of glutamate decarboxylase mutant enzyme activity:

[0035] S1. The recombinant bacteria L100 constructed in Example 2, the wild-type strain W100 constructed in Comparative Example 1, and the glutamate decarboxylase mutant G100 constructed in Comparative Example 2 were inoculated into 3 mL of LB medium containing Amp, cultured at 37 ° C with shaking overnight, and transferred to 50 mL of fresh LB medium at a 3% inoculum the next day, and cultured at 37 ° C, 180 rpm until OD 600 =0.6, IPTG was added to a final concentration of 0.4 mmol / L for induction, and the induction was carried out at 25°C and 180 rpm for 22 h;

[0036] S2. Centrifuge the bacterial suspension at 45,000 rpm for 15 min, discard the supernatant, add 15 mL of lysis buffer (pH 7.5, 20 mmol / L Tris-HCl buffer, 200 mmol / L NaCl), resuspend the cells, and sonicate for 20 min. Centrifuge at 5,000 rpm for 10 min at 4°C to obtain the crude extracellular enzyme solution.

[0037] S3. The crude enzyme solution was separated and purified using a Ni-NTA affinity chromatography column. After loading, washing, and elution, the eluate containing the target protein was collected and ultrafiltration was performed to remove small molecules to obtain the pure enzyme. The pure enzyme concentration was then determined. Wash buffer (pH 7.5): 20 mmol / L Tris-HCl buffer, 200 mmol / L NaCl, and 50 mmol / L imidazole.

[0038] Elution buffer (pH 7.5): 20 mmol / L Tris-HCl buffer, 200 mmol / L NaCl, 500 mmol / L imidazole;

[0039] Protein storage buffer (pH 7.5): 50 mmol / L sodium dihydrogen phosphate-citrate buffer, 200 mmol / L NaCl;

[0040] S4. Glutamate decarboxylase activity assay: Using the colorimetric method, 3 mL of substrate solution (pH 4.5 sodium hydrogen phosphate-citrate buffer containing 50 mmol / L sodium L-glutamate and 0.1 mmol / L PLP) and 10 μL of crude enzyme solution were reacted at 50°C for a certain period of time, and then the reaction was terminated by boiling in a water bath for 3 minutes. 400 μL of the reaction solution was added with 100 μL of sodium carbonate buffer (1 mol / L), 500 μL of boric acid buffer (0.2 mol / L, pH 9.0), 1.0 mL of phenol (6%), and 1.0 mL of sodium hypochlorite solution (effective chlorine concentration greater than 7.5). The mixture was allowed to stand for 6 minutes, and then boiled in a water bath for 10 minutes. The solution was immediately cooled in an ice bath for 10 minutes. 2 mL of 60% ethanol was added until color was developed, and the absorbance value A was measured. 640 .

[0041] Enzyme activity definition: The enzyme activity unit (U) is defined as the amount of enzyme required to produce 1 μmol of GABA per minute under the assay conditions.

[0042] 2. pH stability determination of glutamate decarboxylase mutants:

[0043] The enzyme was stored in sodium hydrogen phosphate-citrate buffer at pH 5.0, 5.5, 6.0, 6.5, and 7.0, respectively. After being kept in a 30°C water bath for 3 h, the residual enzyme activity was measured at pH 5.0 and 50°C according to the above enzyme activity determination method to investigate the pH stability of the enzyme.

[0044] 3. Determination of temperature stability of glutamate decarboxylase mutants:

[0045] The enzyme stored in sodium hydrogen phosphate-citrate buffer at pH 5.0 was incubated in a water bath at 30°C, 35°C, 40°C, 45°C, 50°C, and 55°C, respectively. The total incubation time was set to 6 h. The residual enzyme activity was determined at pH 5.0 and 50°C according to the above enzyme activity determination method.

[0046] The results of enzyme activity determination of glutamate decarboxylase mutants and wild types showed that the specific enzyme activity of glutamate decarboxylase expressed by glutamate decarboxylase mutant L100 was 183.55 U / mg, which was 50.03% higher than that of wild-type glutamate decarboxylase W100 (122.34 U / mg) and 4.1% higher than that of glutamate decarboxylase mutant G100 in which histidine H at position 467 was mutated to serine S (specific enzyme activity 176.31 U / mg).

[0047] Table 1 Effect of different pH on glutamate decarboxylase activity

[0048]

[0049] As shown in Table 1, the enzyme activity of the glutamate decarboxylase mutant L100 is the highest in the appropriate pH range (5-6). Under the optimal pH (5.0), the enzyme activity of the glutamate decarboxylase mutant L100 is 29% higher than that of the wild type and 9.3% higher than that of the glutamate decarboxylase mutant G100.

[0050] Table 2 Effects of different temperatures on glutamate decarboxylase activity

[0051]

[0052] As shown in Table 2, the enzyme activity of the glutamate decarboxylase mutant L100 was 45.9% after being incubated at a non-optimal reaction temperature (50° C.) for 6 h, which was 40.8% higher than that of the wild type and 18.6% higher than that of the glutamate decarboxylase mutant G100.

[0053] SEQ ID NO: 1

[0054] MPQWHPHREQKNLPDEFPVNPLFSRQGEVTIPRLRIGDQGMLPETAYQIIHDEI

[0055] ALDGNARLNLATFVTTTWMEPDAKRLYGESFDKNMIDKDEYPQTAAIEERCV

[0056] RILADLWNSPNPDTTMGVSTTGSSEACMLGGLALKRRWQKLRKSKGLSTDR

[0057] PNIVFSSSVQVVWEKFANYWDVEPRYVNINPDHPYLDAEGVINAVDENTIGV

[0058] VPILGVTYTGGYEPIAAIAKALDELQEKTGLDIPIHVDAASGGFIAPFLQPDLIW

[0059] DFRLPRVKSINVSGHKYGLVYPGLGWVIWREKEDLPEDLIFRVSYLGGNMPT

[0060] FALNFSRPGAQVLLQYYNFLRLGKDGYYAVQKTSQENALFLSKEIGEMDAFE

[0061] ILADGSDIPVLAWKLKEDYTPNWTLYDLSRQLRTYGWQVPAYPLPADMEEIT

[0062] IMRIVVRNGFSRDLAHLFMVNFKQAVEFLNSLDRPVLKDTKYDNGFHH

[0063] SEQ ID NO:2

[0064] ATGCCTCAATGGCATCCGCATCGTGAACAAAAAAATTTACCTGATGAATTTC

[0065] CTGTTAATCCGCTTTTTTCTCGACAAGGAGAAGTGACAATTCCAAGACTGC

[0066] GTATCGGTGATCAAGGTATGCTTCCGGAAACGGCTTATCAAATCATTCATGA

[0067] CGAAATTGCTTTAGACGGAAATGCCCGCTTGAATTTAGCTACGTTTGTTACT

[0068] ACGTGGATGGAGCCTGATGCAAAGCGTTTGTACGGAGAATCTTTTGATAAA

[0069] AATATGATCGATAAAGATGAGTATCCGCAGACAGCGGCTATTGAAGAGAGA

[0070] TGTGTACGTATTTTAGCGGATTTGTGGAATTCACCTAATCCTGATACCACGAT

[0071] GGGCGTTTCTACTACAGGTTCATCTGAAGCATGTATGCTTGGTGGACTAGCG

[0072] TTAAAAAGACGATGGCAGAAACTGCGTAAAAGTAAAGGGCTATCAACGGA

[0073] CCGCCCCAATATTGTATTTAGTTCATCGGTTCAAGTGGTATGGGAGAAGTTC

[0074] GCAAACTATTGGGACGTAGAGCCTCGTTATGTGAATATTAATCCAGATCATC

[0075] CTTATTTAGATGCAGAAGGCGTGATTAATGCGGTTGACGAAAATACAATTGG

[0076] CGTCGTACCGATTCTTGGAGTCACGTATACAGGGGGTTACGAACCAATAGC

[0077] TGCTATCGCAAAAGCATTAGATGAGTTACAGGAAAAAACAGGGTTGGATAT

[0078] TCCTATCCATGTAGATGCTGCTTCTGGAGGTTTTATCGCTCCATTTCTTCAAC

[0079] CAGACCTTATCTGGGATTTCCGCTTGCCGCGAGTAAAGTCCATTAACGTGTC

[0080] AGGACACAAGTATGGTTTAGTTTACCCTGGCTTGGGATGGGTGATTTGGAG

[0081] AGAAAAAGAGGACTTGCCTGAAGATCTTATTTTCCGCGTTTCTTATTTAGGG

[0082] GGCAACATGCCAACTTTTGCGCTCAACTTCTCTAGACCAGGAGCACAAGTC

[0083] CTTTTGCAGTACTACAATTTCTTGCGTTTAGGTAAAGACGGCTATTATGCCG

[0084] TGCAAAAAACCTCCCAAGAAAACGCGCTGTTTCTTAGCAAAGAAATTGGA

[0085] GAAATGGACGCATTCGAAATTCTTGCTGATGGTTCAGATATCCCGGTTCTTG

[0086] CTTGGAAACTGAAAGAAGACTATACACCAACTGGACTCTTTATGATTTGT

[0087] CTAGACAACTGCGTACGTACGGATGGCAAGTTCCTGCTTACCCACTCCCAG

[0088] CAGACATGGAAGAAATCACAATCATGCGCATTGTTGTTAGAAATGGGTTTT

[0089] CAAGAGACCTTGCTCATTTATTTATGGTTAATTTCAAACAAGCCGTTGAATT

[0090] TCTTAACTCGTTGGATAGACCTGTTCTTAAAGACACGAAATACGACAATGG

[0091] ATTTACGCATTAA

[0092] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any person skilled in the art can, without departing from the spirit and technical solution of the present invention, use the methods and technical contents disclosed above to make many possible changes and modifications to the technical solution of the present invention, or modify it into an equivalent embodiment with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A highly active glutamate decarboxylase mutant, characterized in that: The glutamate decarboxylase mutant is obtained by performing site-directed mutagenesis on the wild-type glutamate decarboxylase derived from Bacillus megaterium, specifically, the histidine at position 466 of the wild-type glutamate decarboxylase is mutated to threonine; the amino acid sequence of the wild-type glutamate decarboxylase is shown in SEQ ID NO:

1.

2. The glutamate decarboxylase mutant according to claim 1, wherein: The nucleotide sequence encoding the glutamate decarboxylase mutant is shown in SEQ ID NO:

2.

3. A recombinant expression vector, characterized in that: The recombinant expression vector contains the glutamate decarboxylase mutant according to claim 1 or the nucleotide sequence according to claim 2.

4. The recombinant expression vector according to claim 3, characterized in that: The recombinant expression vector is a prokaryotic expression vector, including but not limited to pET21a and pXMJ19.

5. A recombinant cell, characterized in that: The recombinant cell contains the glutamate decarboxylase mutant according to claim 1 or the nucleotide sequence according to claim 2 or the recombinant expression vector according to claim 3 or 4.

6. The recombinant cell according to claim 5, wherein: The recombinant cells are prokaryotic cells, including but not limited to Escherichia coli and Corynebacterium glutamicum.

7. Use of the glutamate decarboxylase mutant according to claim 1, the nucleotide sequence according to claim 2, the recombinant expression vector according to claim 3 or 4, or the recombinant cell according to claim 5 or 6 in the biosynthesis of γ-aminobutyric acid.

8. The use according to claim 7, characterized in that: The method comprises the steps of preparing the glutamate decarboxylase mutant or nucleotide sequence or recombinant expression vector or recombinant cell to produce gamma-aminobutyric acid, and collecting the produced gamma-aminobutyric acid.

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