Glutamate decarboxylase mutant and application thereof in preparation of gamma-aminobutyric acid

Abstract

The invention relates to the technical field of biology, in particular to a glutamate decarboxylase mutant and application thereof in preparation of gamma-aminobutyric acid. The amino acid sequence of the glutamate decarboxylase mutant provided by the invention is obtained by mutating at least one of the following positions of the amino acid sequence of wild-type glutamate decarboxylase: the 278th position, the 282nd position, the 285th position and the 405th position, and the amino acid sequence of the wild-type glutamate decarboxylase is shown as SEQ ID NO: 1. According to the mutant provided by the invention, the enzyme activity of glutamate decarboxylase is improved, the thermal stability is enhanced, and the industrial requirements of efficient and low-cost preparation of gamma-aminobutyric acid are met.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a glutamate decarboxylase mutant and its application in the preparation of γ-aminobutyric acid. Background Technology

[0002] Gamma-aminobutyric acid (GABA) is an important four-carbon non-protein amino acid. As a major inhibitory neurotransmitter in the mammalian central nervous system, it possesses various physiological functions, including lowering blood pressure, reducing anxiety, and improving sleep. It has been widely used in medicine, food, feed, and chemical industries. Furthermore, GABA can serve as a precursor for the synthesis of bio-based polymers (such as nylon 4). Therefore, developing efficient and low-cost GABA production processes is of great significance.

[0003] Currently, the biocatalytic method using glutamate decarboxylase (GAD) to catalyze the decarboxylation of L-glutamate (L-Glu) to produce GABA has attracted much attention due to its mild conditions and high specificity. However, naturally derived GADs often suffer from low enzyme activity, poor thermal stability, and instability under the acidic conditions required for industrial applications, which limits their industrial application.

[0004] To overcome these shortcomings, researchers have attempted protein engineering of GAD. Takagi et al. (ChemBioChem, 2022, 23, e202100447) designed an artificial enzyme called FcGAD based on the homologous sequence of GAD derived from Lactobacillus using a consensus-based design method. This FcGAD exhibits high expression levels in E. coli and demonstrates superior acid tolerance and thermal stability compared to natural GAD, with an optimal pH of 3.5 and an optimal temperature of 70°C. However, there is still room for further improvement in the catalytic efficiency of FcGAD to reduce the cost and cycle time of industrial production.

[0005] Therefore, in this field, further modifications are still needed to existing GADs (especially engineered enzymes with improved performance such as FcGAD) to obtain mutants with higher catalytic activity and better stability to meet the needs of industrial production of GABA. Summary of the Invention

[0006] This invention aims to at least partially solve one of the technical problems in the related art. Therefore, one object of this invention is to provide a mutant of glutamate decarboxylase, which exhibits significantly enhanced enzyme activity, high catalytic efficiency, and improved thermal stability, meeting the industrial demand for efficient and low-cost preparation of γ-aminobutyric acid (GABA).

[0007] Therefore, a first aspect of the present invention provides a mutant of glutamate decarboxylase. According to an embodiment of the present invention, the amino acid sequence of the mutant glutamate decarboxylase is obtained by mutating at least one of the following positions in the amino acid sequence of wild-type glutamate decarboxylase: The 278th, 282nd, 285th, and 405th positions, The amino acid sequence of the wild-type glutamate decarboxylase is shown in SEQ ID NO: 1.

[0008] This invention obtains a mutant of glutamate decarboxylase by site-directed mutagenesis of at least one of the amino acid positions 278, 282, 285 and 405 relative to the wild-type glutamate decarboxylase. This mutant improves the enzyme activity and thermostability of glutamate decarboxylase.

[0009] According to an embodiment of the present invention, compared with the amino acid sequence of wild-type glutamate decarboxylase, the amino acid sequence of the mutant glutamate decarboxylase has at least one of the following mutations (1)-(4): (1) Tyrosine at position 278 is mutated to phenylalanine; (2) Tyrosine at position 282 is mutated to phenylalanine; (3) The isoleucine at position 285 is mutated into valine or leucine; (4) Proline at position 405 is mutated to threonine.

[0010] This invention modifies the glutamate decarboxylase FcGAD (amino acid sequence shown in SEQ ID NO: 1) from Lactobacillus brevis by site-directed mutagenesis at specific positions, resulting in a mutant that improves the enzyme activity and thermostability of the glutamate decarboxylase.

[0011] According to an embodiment of the present invention, compared with the amino acid sequence of wild-type glutamate decarboxylase, the amino acid sequence of the mutant glutamate decarboxylase has the following 1)-10) mutations: 1) Tyrosine at position 278 is mutated to phenylalanine; or 2) Tyrosine at position 282 is mutated to phenylalanine; or 3) The isoleucine at position 285 is mutated to valine; or 4) The isoleucine at position 285 is mutated to leucine; or 5) The proline at position 405 is mutated to threonine; or 6) Tyrosine at position 278 is mutated to phenylalanine, and isoleucine at position 285 is mutated to valine; or 7) Tyrosine at position 282 is mutated to phenylalanine, and isoleucine at position 285 is mutated to valine; or 8) The isoleucine at position 285 is mutated to valine, and the proline at position 405 is mutated to threonine; or 9) Tyrosine at position 278 is mutated to phenylalanine, and isoleucine at position 285 is mutated to leucine; or 10) Tyrosine at position 278 is mutated to phenylalanine, isoleucine at position 285 is mutated to valine, and proline at position 405 is mutated to threonine.

[0012] According to a preferred embodiment of the present invention, the mutant of glutamate decarboxylase, based on the amino acid sequence of wild-type glutamate decarboxylase, has the mutations described in 1)-10) above, which can further enhance the enzyme activity catalyzing the decarboxylation of L-glutamate (L-Glu) to generate GABA, enhance thermal stability, and further reduce the production cost of industrial production of GABA.

[0013] A second aspect of the present invention provides an isolated nucleic acid. According to an embodiment of the present invention, the nucleic acid encodes a mutant of the glutamate decarboxylase described in the first aspect.

[0014] A third aspect of the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector comprises the isolated nucleic acid described in the second aspect.

[0015] A fourth aspect of the present invention provides a host cell. According to an embodiment of the present invention, the host cell comprises the expression vector described in the second aspect.

[0016] The fifth aspect of this invention provides the use of the mutant of glutamate decarboxylase described in the first aspect, the isolated nucleic acid described in the second aspect, the expression vector described in the third aspect, and the host cell described in the fourth aspect in the preparation of γ-aminobutyric acid.

[0017] The purpose of this invention is to provide a glutamate decarboxylase mutant with higher catalytic efficiency and better thermal stability to solve the problem of insufficient catalytic activity of GAD enzymes in the prior art. To this end, this invention provides a glutamate decarboxylase mutant, which is generated by mutating at least one amino acid site selected from the following amino acid sites or combinations thereof, starting from the amino acid sequence shown in SEQ ID NO: 1: tyrosine at position 278 is mutated to phenylalanine, tyrosine at position 282 is mutated to phenylalanine, isoleucine at position 285 is mutated to valine or leucine, and proline at position 405 is mutated to threonine; and the mutant possesses glutamate decarboxylase activity. Using the glutamate decarboxylase mutant provided by this invention, enzyme activity is significantly improved, catalytic efficiency is high, and thermal stability is enhanced, meeting the industrial demand for efficient and low-cost preparation of γ-aminobutyric acid.

[0018] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0019] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 The three-dimensional spatial structure of the protein obtained by protein modeling of FcGAD using software tools is shown. Figure 2 The results of SDS-PAGE gel electrophoresis of the supernatant and precipitate obtained after culturing the mutant B1-B12 genetically engineered bacteria are shown. Figure 3 The results of SDS-PAGE gel electrophoresis of the supernatant and precipitate obtained after culturing the double mutant B13-B16 genetically engineered bacteria are shown, with B5 genetically engineered bacteria as a control. Figure 4 The results of SDS-PAGE gel electrophoresis of the supernatant and precipitate obtained after culturing the triple mutant B17 genetically engineered bacteria are shown, with the B15 genetically engineered bacteria as a control. Detailed Implementation

[0020] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0021] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0022] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0023] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0024] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0025] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0026] According to a specific embodiment of the present invention, the present invention provides a mutant of glutamate decarboxylase, the amino acid sequence of which is obtained by mutating at least one of the following positions in the amino acid sequence of wild-type glutamate decarboxylase: The 278th, 282nd, 285th, and 405th positions, The amino acid sequence of the wild-type glutamate decarboxylase is shown in SEQ ID NO: 1.

[0027] SEQ ID NO: 1 is shown below: MLYGKKNREEEEYLTPIFGSSAEGEDLPKYKLNKESIEPRIAYRLVKDQLLDEGNARLNLATFCQTYMEPEATKLMAETLEKNAIDKSEYPRTAEIENRCVNIIADLWHAPKDEKF LGTSTVGSSEACLMLGGMAMKFAWRNRAEKLGLDINAKKPNLVISSGYQVCWEKFCVYWDIEMRTVPMDEDHMSLNVDKVLDYVDEYTIGIVGILGITYTGKYDDIKALDDLVEKYNQ TTDYKVYIHVDAASGGFFTPFVEPELEWDFRLKNVISINTSGHKYGLVYPGIGWVLWRDQEYLPKELIFKVSYLGGEMPTMAINFSRSASQIIGQYYNFLRFGFEGYREIHERTHDV ALYLAKELEKTGLFEIYNDGSNLPIVCYKLKEDANVKWTLYDLADRLLMKGWQVPAYPLPKNLDDIIIQRIVCRADLGMNMAEEFIEDFKTAINELNNAHILFHKEEEKKKYGFTH* According to a specific embodiment of the present invention, compared with the amino acid sequence of wild-type glutamate decarboxylase, the amino acid sequence of the mutant glutamate decarboxylase has at least one of the following mutations (1)-(4): (1) Tyrosine at position 278 is mutated to phenylalanine; (2) Tyrosine at position 282 is mutated to phenylalanine; (3) The isoleucine at position 285 is mutated into valine or leucine; (4) Proline at position 405 is mutated to threonine.

[0028] According to a specific embodiment of the present invention, compared with the amino acid sequence of wild-type glutamate decarboxylase, the amino acid sequence of the mutant glutamate decarboxylase has a mutation of tyrosine at position 278 to phenylalanine; or a mutation of tyrosine at position 282 to phenylalanine; or a mutation of isoleucine at position 285 to valine or leucine; or a mutation of proline at position 405 to threonine; or any combination of the above single-point mutations, such as any two-position mutation or three-position mutation, all of which are covered within the protection scope of the mutant glutamate decarboxylase of the present invention.

[0029] According to a specific embodiment of the present invention, compared with the amino acid sequence of wild-type glutamate decarboxylase, the amino acid sequence of the mutant glutamate decarboxylase has the following 1)-10) mutations: 1) Tyrosine at position 278 is mutated to phenylalanine; or 2) Tyrosine at position 282 is mutated to phenylalanine; or 3) The isoleucine at position 285 is mutated to valine; or 4) The isoleucine at position 285 is mutated to leucine; or 5) The proline at position 405 is mutated to threonine; or 6) Tyrosine at position 278 is mutated to phenylalanine, and isoleucine at position 285 is mutated to valine; or 7) Tyrosine at position 282 is mutated to phenylalanine, and isoleucine at position 285 is mutated to valine; or 8) The isoleucine at position 285 is mutated to valine, and the proline at position 405 is mutated to threonine; or 9) Tyrosine at position 278 is mutated to phenylalanine, and isoleucine at position 285 is mutated to leucine; or 10) Tyrosine at position 278 is mutated to phenylalanine, isoleucine at position 285 is mutated to valine, and proline at position 405 is mutated to threonine.

[0030] According to a specific embodiment of the present invention, the present invention provides a nucleic acid sequence encoding the mutant of the glutamate decarboxylase.

[0031] According to a specific embodiment of the present invention, the present invention provides a recombinant vector containing a nucleic acid sequence encoding a mutant of the aforementioned glutamate decarboxylase, and a genetically engineered bacterium containing a nucleic acid sequence encoding the mutant of the aforementioned glutamate decarboxylase. Specifically, the vector can be any of various expression vectors, including but not limited to any one of the following: pET expression vector, pCW expression vector, pUC expression vector, or pPIC9k expression vector. The host cell of the genetically engineered bacterium can be any suitable host cell, including but not limited to Escherichia coli, Bacillus subtilis, Streptomyces, or Pichia pastoris.

[0032] The present invention also relates to mutants of the glutamate decarboxylase, nucleic acids encoding mutants of glutamate decarboxylase, expression vectors containing the nucleic acids, and the use of the genetically engineered bacteria in the preparation of γ-aminobutyric acid.

[0033] It should be noted that there are no particular limitations on the conditions for preparing γ-aminobutyric acid using mutant glutamate decarboxylase; the catalytic conditions can be those commonly used in the field of enzyme catalysis. However, regardless of whether optimized or conventional reaction conditions are used, the mutant glutamate decarboxylase of this invention exhibits better catalytic activity, higher catalytic efficiency, and improved thermal stability compared to the mutant wild-type glutamate decarboxylase.

[0034] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0035] Materials and methods 1) Materials: Plasmid pET15b was purchased from Changsha Aibiwei Biotechnology Co., Ltd.; restriction endonucleases such as NdeI and BamHI, high-fidelity enzyme premix, and one-step rapid cloning kit were purchased from Shanghai Yisheng Biotechnology Co., Ltd.; synthesized primers, E. coli BL21(DE3) competent cells, DNA marker, plasmid extraction kit, DNA gel purification kit, ampicillin, and isopropyl-β-d-thiogalactoside were all purchased from Shanghai Sangon Biotech Co., Ltd.; all chemical reagents were of analytical grade from Sinopharm. The plasmid extraction procedure followed the instructions for the plasmid extraction kit; the DNA gel purification procedure followed the instructions for the DNA gel purification kit; and the DNA fragment ligation procedure followed the instructions for the one-step rapid cloning kit. 2) LB medium (g / L): 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, sterilized at 121℃ for 20 min; 3) Glutamate decarboxylase substrate reaction solution: 200 mM L-glutamic acid, 0.1 mM pyridoxal phosphate (PLP), pH 3.5; 4) Determination of enzyme activity: Accurately measure the supernatant of the ultrasonically disrupted bacterial solution and add it to the substrate reaction solution preheated to 50℃ to make the final concentration of the bacterial cells 5 mg / mL. Then, stir the reaction in a 50℃ water bath for 30 min, add an equal volume of 10% hydrochloric acid solution to terminate the reaction, shake well and centrifuge, and take the supernatant for HPLC analysis, using L-glutamic acid and γ-aminobutyric acid standards as controls. 5) HPLC analysis method: Thermo Fisher Scientific ODS-2 Hypersil™ (4.6 × 250 mm, 5 µm); flow rate 1.0 mL / min; detection wavelength 190 nm; mobile phase for glutamate decarboxylase reaction: methanol: 0.05 mol / L potassium dihydrogen phosphate + 0.005 mol / L sodium decane sulfonate (pH 2.30) = 7:93; 6) Definition of enzyme activity unit: Under the conditions of pH 3.5 and temperature 50℃, the amount of enzyme required to produce 1 micromole (μmol) of γ-aminobutyric acid per unit time is defined as 1U.

[0036] Example 1: Construction of wild-type glutamate decarboxylase FcGAD genetically engineered bacteria (1) Wild-type FcGAD gene synthesis The amino acid sequence of the glutamate decarboxylase FcGAD derived from Lactobacillus brevis is shown in SEQ ID NO: 1. Based on the codon bias of Escherichia coli, the gene coding sequence was codon optimized. NdeI and BamHI restriction sites were added to both ends of the coding sequence, and the resulting FcGAD (SEQ ID NO: 2) was synthesized artificially by a biotechnology company.

[0037] The amino acid sequence of the glutamate decarboxylase FcGAD is shown in SEQ ID NO: 1: MLYGKKNREEEEYLTPIFGSSAEGEDLPKYKLNKESIEPRIAYRLVKDQLLDEGNARLNLATFCQTYMEPEATKLMAETLEKNAIDKSEYPRTAEIENRCVNIIADLWHAPKDEKFLGTSTVGSSEACMLGGMAMKFAWRNRAEKLGLDINAKKPNLVISSGYQVCWEKFCVYWDIEMRTVPMDEDHMSLNVDKVLDYVDEYTIGIVGILGITYTGKYDDIKALDDLVEKYNQTTDYKVYIHVDAASGGFFTPFVEPELEWDFRLKNVISINTSGHKYGLVYPGIGWVLWRDQEYLPKELIFKVSYLGGEMPTMAINFSRSASQIIGQYYNFLRFGFEGYREIHERTHDVALYLAKELEKTGLFEIYNDGSNLPIVCYKLKEDANVKWTLYDLADRLLMKGWQVPAYPLPKNLDDIIIQRIVCRADLGMNMAEEFIEDFKTAINELNNAHILFHKEEEKKKYGFTH* The sequence after codon optimization of the FcGAD gene coding sequence, as shown in SEQ ID NO: 2: (2) Construction of wild-type FcGAD-pET15b vector FcGAD was constructed into the cloning site of the E. coli expression vector pET15b. Primers for vector construction were designed using 1-F (5'-ggtttcacccactaactcgaggatccggctg-3', as shown in SEQ ID NO: 3) / 1-R (5'-ttccatatagcatatggctgccgcgcggcac-3', as shown in SEQ ID NO: 4) as primers, with the purchased vector pET15b as a template, and PCR amplification was performed using 2×Hieff Canace® Plus PCR Master Mix (With Dye) high-fidelity enzyme premix. Primers 2-F (5'-atatgctatatggaaaaaagaatagggaggaa-3', as shown in SEQ ID NO: 5) / 2-R (5'-cagccggatcctcgagttagtgggtgaaacc-3', as shown in SEQ ID NO: 6) were used to amplify the FcGAD gene DNA fragment (SEQ ID NO: 3) synthesized by the biotechnology company. 2) Using a high-fidelity enzyme premix as a template, PCR amplification was performed to obtain FcGAD gene DNA fragments with 5' and 3' ends completely identical to the end sequences of the linearized vector. The fragments were detected by 1% agarose gel electrophoresis, and the amplified linearized vector (5.7 kb) and FcGAD gene (1.4 kb) DNA fragments were recovered using a DNA gel purification kit. Homologous recombination of the gel-recovered linearized vector and FcGAD gene fragments was then performed using the Hieff Clone® Plus One Step Cloning Kit. The recombination system consisted of: 2.5 μL of pET15b linearized vector, 2.5 μL of FcGAD gene fragment, and 5 μL of 2×Hieff Clone® Enzyme Premix. The reaction was carried out at 50°C for 30 min.

[0038] (3) Construction of wild-type FcGAD genetically engineered bacteria The homologous recombination product obtained in (2) was transformed into E. coli BL21(DE3) competent cells by heat shock. The transformation product was plated on LB agar plates containing 50 μg / mL ampicillin and incubated overnight at 37°C. Positive transformants were selected and sent to a gene company for sequencing. Transformants with correct sequencing results contained wild-type cells. FcGAD The gene plasmid vector FcGAD-pET15b is a transformant that is also a wild-type FcGAD genetically engineered bacterium.

[0039] Example 2: Determination of a single site-directed mutation site in FcGAD To obtain the three-dimensional structural information of FcGAD, a homology modeling method based on sequence similarity was used for prediction. Based on sequence consistency, coverage, and structural quality indicators such as template resolution and R-factor, the model was ultimately selected from... Lactobacillus brevis CGMCC 1306 (PDB ID: 5GP4) was used as the modeling template, and PyMol was used to visualize and process the obtained model. The model was evaluated through stereochemical quality analysis (Ramachandran plot analysis) and three-dimensional profile-sequence compatibility analysis. The results are as follows: Figure 1 As shown, 97% of the residues are located within the allowed region, while only 0.2% (located in the highly flexible loop region) are located within the disallowed region, indicating excellent skeletal structure. Based on literature reports, the FcGAD structure is a dimer, and in crystals it is a trimer (due to crystal stacking). Each monomer contains a PLP-binding domain and a small structural domain. Residues such as S124, S128, C171, I212, and S274 stabilize the PLP through hydrogen bonding and hydrophobic interactions. The loop Y306-E312 is located at the active site entrance and is crucial for the enzyme's catalytic activity. Molecular docking was performed using the glide-dock program in Schrodinger Suites software, and the results showed that the substrate interacts with residues such as R420, H276, and K277. Therefore, in order to improve the enzyme activity of FcGAD, we mainly screened the substrate, PLP, and residues within 5 Å around the LOOP loop (Y306-E312), and predicted the change in free energy after mutation using the ProStab online website. A negative ΔΔG value indicates that the mutation makes the protein more stable, which is beneficial to the improvement of activity. The mutation sites are shown in Table 1 below.

[0040] Table 1:

[0041] Example 3: Preparation and expression of FcGAD single mutant Using the original plasmid as a template, site-directed mutagenesis was performed to obtain the recombinant plasmid FcGAD-X-pET15b carrying a glutamate decarboxylase mutant. The site-directed mutagenesis method is as follows: The primer sequences used to design and obtain mutants are shown in Table 2 (the bolded parts in the primer sequences are the bases corresponding to the mutation sites).

[0042] Table 2:

[0043] Using G275A-F / 2-R primers and FcGAD-pET15b plasmid as a template, a small gene fragment with the G275A mutation site was amplified by PCR with 2×Hieff Canace® Plus PCR Master Mix (With Dye) high-fidelity enzyme premix. Using 2-F / G275A-R primers and FcGAD-pET15b plasmid as a template, a large DNA fragment with identical 5' and 3' ends to the small gene fragment was amplified by PCR with the same sequence at both ends. The amplified DNA fragments were detected by 1% agarose gel electrophoresis and recovered using a DNA gel purification kit. Homologous recombination of the recovered DNA fragments was performed using the Hieff Clone® Plus One Step Cloning Kit. The recombination system consisted of 2.5 μL of the large DNA fragment, 2.5 μL of the small DNA fragment, and 5 μL of 2×Hieff Clone® Enzyme Premix. The reaction was carried out at 50°C for 30 min.

[0044] The obtained homologous recombination product was transformed into Escherichia coli BL21(DE3) competent cells by heat shock. The transformation product was plated on LB agar plates containing 50 μg / mL ampicillin and cultured overnight at 37°C. Positive transformants were selected and sent to a gene company for sequencing. The correctly sequenced transformant was the plasmid vector FcGAD-G275A-pET15b containing the single mutant FcGAD gene. This transformant was also a single mutant FcGAD-G275A genetically engineered bacterium, numbered B1. The following engineered bacteria were constructed in the same manner: B2 with the Y278F mutation, B3 with the Y282F mutation, B4 with the P283L mutation, B5 with the I285V mutation, B6 with the I285L mutation, B7 with the D175A mutation, B8 with the I212L mutation, B9 with the P405T mutation, B10 with the I418V mutation, B11 with the Q419L mutation, and B12 with the Q419V mutation.

[0045] The correctly sequenced mutant B1-B12 genetically engineered bacteria were inoculated into test tubes containing 4 mL of LB medium (containing 50 μg / mL ampicillin). After overnight incubation at 37°C and 200 rpm, the cultures were transferred to 50 mL of LB medium and incubated at 37°C and 200 rpm until OD (outlet count) was reached. 600The concentration was initially set at 0.6-0.8, and IPTG was added to a final concentration of 0.2 mM. The mixture was incubated at 20℃ and 200 rpm for 18 h. After centrifugation at 9000 rpm for 5 min, the supernatant was removed, and the bacterial cell pellet was collected. The pellet was resuspended in water to a concentration of 100 mg / mL, sonicated for 10 min, and the pellet was removed by centrifugation. The supernatant was then analyzed by SDS-PAGE gel electrophoresis. The results are as follows: Figure 2 As shown, this indicates that all of these mutant proteins are expressed normally.

[0046] Example 4: Enzyme activity assay of FcGAD single mutant Add 25 μL of enzyme solution to a centrifuge tube containing 975 μL of substrate solution (water containing 200 mM L-glutamic acid and 0.1 mM pyridoxal phosphate (PLP)) and incubate at 50℃ for 30 min. After the reaction is complete, add an equal volume of 10% hydrochloric acid solution to terminate the reaction, shake well, centrifuge, and take the supernatant for HPLC analysis, using L-glutamic acid and γ-aminobutyric acid standards as controls.

[0047] The relative enzyme activities of each single mutant are shown in Table 3.

[0048] Table 3:

[0049] The results in Table 3 show that the enzyme activities of mutants containing single-point mutations of Y278F (B2), Y282F (B3), I285V (B5), I285L (B6), and P405T (B9) are significantly increased compared with wild-type enzyme activities. Among them, the enzyme activity of mutant B5 containing I285V is the largest, which is 1.31 times that of wild-type enzyme activity.

[0050] Example 5: Preparation and expression of FcGAD double mutants Five variants were selected from mutation sites showing a significant increase (approximately 20% or more) in enzyme activity compared to the wild-type enzyme. These variants are listed as B2, B3, B5, B6, and B9 in Table 3. The base sequences of the corresponding genes were analyzed, and amino acid mutation information was examined. Combination mutations were then performed and the mutants were expressed. The expression vector construction and preparation methods were the same as in Example 3, resulting in double mutants. The SDS-PAGE gel electrophoresis analysis results are shown below. Figure 3 As shown, this indicates that all these double mutant proteins are expressed normally. The protein expression and relative enzyme activity of the obtained double mutants were determined using the same method as in Example 4, and the results are shown in Table 4.

[0051] Table 4:

[0052] As shown in Table 4, the enzyme activity of the I285V mutants, after being superimposed with Y278F or P405T, or I285L superimposed with Y278F, was further increased compared to the single mutants (B5 or B6), increasing from 1.31 times that of the wild type to 1.39-1.54 times or from 1.19 times to 1.34 times. Among them, the I285V-P405T (B15) enzyme activity increased the most, reaching 1.54 times that of the wild type.

[0053] Example 6: Preparation and expression of FcGAD triple mutant Three double mutants with significantly enhanced enzyme activity prepared in Example 5, namely B13, B15, and B16, were selected. The base sequences of the corresponding genes and amino acid mutation information were analyzed. Combination mutations were then performed and the mutants were expressed. The expression vector construction and preparation methods were the same as in Example 3, resulting in triple mutants. SDS-PAGE gel electrophoresis analysis results are shown in the table below, indicating that the B17 triple mutant protein is expressed normally. The protein expression and relative enzyme activity of the obtained triple mutants were measured using the same method as in Example 4, and the results are shown in Table 5.

[0054] Table 5:

[0055] As shown in Table 5, the enzyme activity of the double mutant containing I285V-P405T, after being superimposed with Y278F, is further improved compared to I285V-P405T (B15), increasing from 1.54 times that of the wild type to 1.67 times.

[0056] Example 7: Determination of the thermostability of FcGAD mutants and wild-type with enhanced enzyme activity The thermal stability of proteins was determined using the supernatant of lysates from nine high-enzyme-activity mutants (B2, B3, B5, B6, B9, B13, and B15-B17) obtained in Examples 4, 5, and 6 above, and WT. The obtained lysate supernatants were treated at 50°C, 55°C, 60°C, 65°C, and 70°C for 30 min, respectively, and then cooled in an ice-water bath. The residual activity was then measured at 50°C. Specific data are shown in Table 6 below.

[0057] Table 6:

[0058] The results showed that among the nine high-enzyme-activity mutants (B2, B3, B5, B6, B9, B13, and B15-B17), five mutants (B2, B5, B13, B16, and B17) containing I285V and Y278F, respectively, exhibited significantly improved thermostability compared to the wild type. Specifically, their activity began to decrease at 65°C, but only decreased by 10-40% at 70°C. However, when I285V or Y278F was combined with P405T for mutation, the thermostability of B15 and B17 decreased slightly, but remained better than the wild type. These examples illustrate that I285V and Y278F have different degrees of effect on improving thermostability.

[0059] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," "some implementations," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0060] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A mutant of glutamate decarboxylase, characterized in that, The amino acid sequence of the mutant glutamate decarboxylase was obtained by mutating at least one of the following positions in the amino acid sequence of the wild-type glutamate decarboxylase: The 278th, 282nd, 285th, and 405th positions, The amino acid sequence of the wild-type glutamate decarboxylase is shown in SEQ ID NO:

1.

2. The mutant according to claim 1, characterized in that, Compared with the amino acid sequence of wild-type glutamate decarboxylase, the amino acid sequence of the mutant glutamate decarboxylase has at least one of the following mutations (1)-(4): (1) Tyrosine at position 278 is mutated to phenylalanine; (2) Tyrosine at position 282 is mutated to phenylalanine; (3) The isoleucine at position 285 is mutated into valine or leucine; (4) Proline at position 405 is mutated to threonine.

3. The mutant according to claim 1, characterized in that, Compared with the amino acid sequence of wild-type glutamate decarboxylase, the amino acid sequence of the mutant glutamate decarboxylase has the following 1)-10) mutations: 1) Tyrosine at position 278 is mutated to phenylalanine; or 2) Tyrosine at position 282 is mutated to phenylalanine; or 3) The isoleucine at position 285 is mutated to valine; or 4) The isoleucine at position 285 is mutated to leucine; or 5) The proline at position 405 is mutated to threonine; or 6) Tyrosine at position 278 is mutated to phenylalanine, and isoleucine at position 285 is mutated to valine; or 7) Tyrosine at position 282 is mutated to phenylalanine, and isoleucine at position 285 is mutated to valine; or 8) The isoleucine at position 285 is mutated to valine, and the proline at position 405 is mutated to threonine; or 9) Tyrosine at position 278 is mutated to phenylalanine, and isoleucine at position 285 is mutated to leucine; or 10) Tyrosine at position 278 is mutated to phenylalanine, isoleucine at position 285 is mutated to valine, and proline at position 405 is mutated to threonine.

4. An isolated nucleic acid, characterized in that, The nucleic acid encodes a mutant of the glutamate decarboxylase according to any one of claims 1-3.

5. An expression carrier, characterized in that, The expression vector comprises the isolated nucleic acid as described in claim 4.

6. A host cell, characterized in that, The host cell comprises the expression vector of claim 5.

7. The use of the mutant of glutamate decarboxylase according to any one of claims 1-3, the isolated nucleic acid according to claim 4, the expression vector according to claim 5, and the host cell according to claim 6 in the preparation of γ-aminobutyric acid.

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