CN111607583A - Glutamic acid decarboxylase mutant, gene and application - Google Patents

Abstract

The invention discloses a glutamic acid decarboxylase mutant, a gene and application. The glutamic acid decarboxylase mutant is obtained by mutating 54 th amino acid residue of wild type glutamic acid decarboxylase from aspartic acid to alanine. The GAD mutant with higher catalytic performance is obtained by modifying the protein by a method for optimizing the surface charge of the protein and combining a site-directed mutagenesis technology. K of mutant D54A compared to wild type_cat/K_mIs 1.80 times of wild type, t_1/214.76 times of wild type, T₅₀ ¹⁵And T_mRespectively increased by 3 compared with the wild type.95 ℃ and 4.80 ℃.

Description

Glutamic acid decarboxylase mutant, gene and application

Technical Field

The invention relates to the technical field of biology, in particular to a glutamic acid decarboxylase mutant, a gene and application.

Background

GABA (gamma-amino butyric acid or gamma-amino butyric acid, GABA for short) is a compound with biological activity, is an important inhibitory nerve transmission substance in the central nervous system of mammals, and has the effects of preventing cancer, relieving cardiovascular diseases, resisting convulsion, lowering blood pressure, tranquilizing and allaying excitement, sobering up and the like. In addition, GABA can be used for synthesizing N-pyrrolidone and nylon 4, and is widely applied in the fields of food, medicine, chemical industry and the like. Glutamate decarboxylase (GAD; EC 4.1.1.15) is a key enzyme for the biological preparation of GABA. In the presence of coenzyme PLP, GAD can specifically catalyze L-glutamic acid (L-Glu) to remove alpha-carboxyl to generate GABA. Because of the disadvantages of low catalytic efficiency and poor stability of natural GAD, the application in industry is limited. Therefore, it is important to improve the catalytic performance of GAD for industrial application.

Most of the protein surface residues are not conserved in the evolution process and have important fine-tuning effect on the catalytic performance of the enzyme. Changes in the charged residues on the surface of the protein may cause changes in the electrostatic field around the active site of the protein, thereby altering the catalytic properties of the enzyme. Research shows that optimizing the surface charge of protein is an effective strategy for improving the catalytic performance of enzyme, and the method is used for improving the catalytic performance by predicting the surface charge-charge interaction energy of the enzyme protein and replacing negatively charged residues on the surface of the protein, which are not beneficial to the catalytic performance of the enzyme, with neutral or positively charged residues. Schweiker et al (Schweiker KL, Zarrine-Afsar A, Davidson AR, actual. computerized design of the Fyn SH3 domain with created static through optimization of surface chargeions[J]Protein science.2007; 16(12) 2694-702.) use TK-SA model to reasonably optimize the surface charge interaction energy of Fyn SH3 domain, successfully improve the stability of the protein, and stabilize the thermal decomposition folding temperature (T) of the mutant_m) 12.3 ℃ higher than the wild type. Zhang et al (Zhang LJ, Tang XM, Cui DB, et al. A method increase protein stability base on the charge-charge interaction, with application to lipase LipK107[ J]Protein science.2014; 23(1):110-6.) through improving TK-SA model algorithm, a set of new design algorithm-ETSS is issued, the ETSS algorithm is used for predicting 4 residues (D113, D149, D213 and D253) which are positioned on the surface of lipase Lipk107 and are crucial to the stability of the lipase, amino acids at 4 sites are replaced by acidic or neutral amino acids to obtain 4 Lipk107 mutants (D113A, D149K, D213A and D253A), and research results show that the half lives (t 113, T113A, D149K, D213A and D253A) of the mutant D113, the mutant D149K, the mutant D213A and the mutant D253A at 50 ℃ are shown_1/2) 12 times, 14 times, 4.5 times and 6 times of the wild type respectively, and the enzyme activity of the mutant D253A is 1.2 times of the wild type. Tu et al (Tu T, Luo H, Meng K, ethyl. improvement in thermal stability of anophe sp. strain Xz8endo olygalacturonase a the optimization of charge-charge interactions [ J]Applied and Environmental microbiology.2015; 81(19) 6938-44) the total interaction energy (E) between charged amino acids i and j of polygalacturonase was calculated using the ETSS program_ij) Deducing 9 residues influencing the stability of the zymoprotein, and constructing 9 mutants by utilizing site-directed mutagenesis, wherein researches show that the mutants D244A and D299R show better thermal stability; semi-inactivation temperature (T) of double mutant D244A/D299R₅₀) And T_mIncreased t at 50 ℃ and 55 ℃ by 17 ℃ and 10.2 ℃ respectively over the wild type_1/2Respectively extending 8.4h and 45min compared with the wild type.

The inventor obtains a mutant strain Lactobacillus brevis (with the preservation number of CGMCC NO.1306) with high GABA yield in earlier stage (yellow handsome. research on the relevant process for preparing gamma-aminobutyric acid by using Lactobacillus brevis. Hangzhou. 2006; Zhejiang university.; Fan E, Huang J, Hu S, Mei L, Yu K.cloning, sequencing and expression of an aggregation decarbonylase gene from the GABA-degrading strain Lactobacillus brevis CGMCC1306.Annals of microbiology.2011; 62(2): 689-98.). Subsequently, the GAD1407 gene was cloned from Lactobacillus brevis Lb.brevis CGMCC No.1306 by molecular biological means and heterologous recombinant expression thereof in E.coli was achieved (Fan E, Huang J, Hu S, Mei L, Yu K.cloning, sequencing and expression of a glutamate-degrading gene from the GABA-producing strain Lactobacillus brevis CGMCC1306. antibiotics of microbiology.2011; 62(2): 689-98.; Lin L, Hu S, Yu K, Huang J, Yao S, Leuti Y, et al. engineering of the Activity of glutamic degradation of Lactobacillus brevis bacterium strain 201412. Ev.2014). In addition, the subject group analyzed the Crystal structure of GAD (PDB ID:5GP4) and provided a theoretical basis for studying the structure-function relationship of GAD (Huang J, Fang H, gaiZC, Mei JQ, Li JN, Hu S, et al.Lactobacillus brevicus CGMCC1306. glutamatedacrylase: Crystal structure and functional analysis. biochemical analysis research communication.2018; 503(3): 1703-9.).

Disclosure of Invention

The invention provides a glutamic acid decarboxylase mutant, a gene and application aiming at the problems of low enzyme activity and poor thermal stability of glutamic acid decarboxylase in the prior art.

A glutamic acid decarboxylase mutant is obtained by mutating 54 th amino acid residue of wild glutamic acid decarboxylase from aspartic acid to alanine.

Preferably, the amino acid sequence of the glutamate decarboxylase mutant is shown as SEQ ID No. 3.

The invention also provides a gene for coding the glutamic acid decarboxylase mutant.

Preferably, the nucleotide sequence of the gene is shown as SEQ ID No. 2. The coding gene of the wild type glutamate decarboxylase is shown as SEQ ID No. 1.

The invention also provides a genetic engineering bacterium containing the gene.

The invention also provides application of the glutamate decarboxylase mutant in catalyzing L-glutamic acid to remove alpha-carboxyl to generate GABA.

The invention also provides application of the gene in catalyzing L-glutamic acid to remove alpha-carboxyl to generate GABA.

The GAD mutant with higher catalytic performance is obtained by modifying the protein by a method for optimizing the surface charge of the protein and combining a site-directed mutagenesis technology. Catalytic efficiency of mutant D54A compared to wild type (k)_cat/K_m) Is 1.80 times of wild type, t_1/214.76 times of wild type, T₅₀ ¹⁵And T_mThe temperature is increased by 3.95 ℃ and 4.80 ℃ respectively compared with the wild type.

Drawings

FIG. 1 is E of each charged residue in GAD_ijAnd (5) value calculation result graph.

FIG. 2 is a SDS-PAGE analysis of wild type and mutant, wherein the lanes are M: protein marker; 1: a wild type; 2: E14A; 3: E14R; 4: D54A; 5: D54K; 6: E114A; 7: E114K.

FIG. 3 is a diagram showing the results of enzyme activity detection of wild type and mutant.

FIG. 4 is a graph showing the results of residual activity assay of wild type and mutant.

FIG. 5 is a graph showing the results of stability tests of the wild type and the mutant, wherein (a) T of the wild type GAD and the mutant D54A₅₀ ¹⁵(ii) a (b) Wild type GAD at 55 ℃ t_1/2(ii) a (c) Mutant D54A t at 55 ℃_1/2(ii) a (d) T of wild type GAD and mutant D54A_m。

FIG. 6 is a graph of the results of MD simulations of wild-type and mutant D54A, wherein (a) the RMSD values of wild-type GAD and mutant D54A under 313K conditions; (b) RMSF values for wild type GAD and mutant D54A under 313K conditions.

Detailed Description

Example 1

(1) Materials and reagents

Brevis CGMCC1306, e.coli DH5 α. PrimeSTAR Max DNA polymerase from TaKaRa; dpn I was purchased from Thermo Scientific; SanPrep column type plasmid DNA small extraction kit, isopropyl-b-D-thiogalactoside (IPTG), Kanamycin sulfate (Kanamycin sulfate), pyridoxal phosphate (PLP), modified Bradford protein concentration determination kit, L-Sodium glutamate hydrate (Sodium glutamate hydrate) from Biotechnology engineering (Shanghai) GmbH; the Ni-NTA chromatography medium is purchased from Beijing Quanjin Biotechnology GmbH; dansyl chloride (DNS-Cl) was purchased from Sigma, Inc. (USA); gamma-aminobutyric acid (gamma-aminobutyric acid) was purchased from Fluka corporation (Switzerland).

(2) Selection of the site of mutation

The total interaction energy (E.sub.d-type) between the i and j points of the wild-type GAD (wild-type) charged amino acid residues was calculated using the ETSS program of the Linux system (version 6.0 of Centos) without taking into account other interactions_ij)，E_ijResidues with a positive value have a negative effect on the overall stability of GAD, while E_ijResidues with negative values have a positive effect on the stability of egg GAD. Theoretically, the change of the charge of the charged residue at a specific position of the protein may cause the change of the surface potential thereof, thereby improving the stability of the protein. Thus, site-directed mutagenesis is used to map to a specific position E_ijThe positive amino acid was mutated to a simple neutral amino acid (alanine) and an amino acid of opposite charge, creating a mutant. All charged amino acid residues were screened according to the following 3 criteria: (1) e_ijResidues with high energy values (positive); (2) residues distal to the GAD active center, (3) residues on the secondary structure of GAD.

Total E between i and j points for the charged amino acid residues of wild-type GAD_ijAs shown in FIG. 1, 149 charged amino acids were found together in GAD, of which 68 residues of E_ijPositive value, 81 residues E_ijThe value is negative. All charged amino acid residues were screened according to 3 criteria, 3 key amino acid residues were screened for GAD catalytic performance (E14, D54 and E114), and subsequently 6 potential mutants were obtained using site-directed mutagenesis techniques (E14A, E14R, D54A, D54K, E114A and E114K).

PCR amplification was performed using the primers in Table 1 and a plasmid containing GAD1407 gene (the gene sequence is shown in SEQ ID No.1, the length is 1407bp, cloned from Lactobacillus brevis Lb. brevis CGMCC No.1306, the acquisition time is 10 months in 2012) as a template. After the PCR product was digested with Dpn I at 37 ℃, it was transferred to E.coli DH 5. alpha. competent cells by chemical transformation, and 1h later the resuscitative solution was spread on LB solid plate containing kanamycin to a final concentration of 50. mu.g/. mu.L to obtain a site-directed mutagenesis library. The recombinant plasmid is sent to Anhui general biosystems, Inc. for nucleotide sequence determination, and the recombinant plasmid with correct sequencing is transformed into E.coli BL21(DE3) competent cells to obtain a target recombinant strain.

TABLE 1 site-directed mutagenesis primers

Remarking: the mutation position is underlined.

(3) Construction of mutant libraries and plasmid extraction

Coli DH5 α competent cells, preserved at-80 ℃, were removed and thawed on ice. 10 μ L of the digest was added to 50 μ L of E.coli DH5 α competent cells, gently mixed by a gun, and left to stand on ice for 30 min. And (3) performing heat shock in a water bath kettle at 42 ℃ for 90s, and quickly cooling the tube on ice for 3-5 min. 600 mul of pre-cooled LB culture medium is added into each tube, and the tubes are revived and cultured for 1h under the conditions of 37 ℃ and 180pm, so that the bacteria are restored to the normal growth state. mu.L of the culture broth was uniformly spread on LB solid medium plates containing Kan at a final concentration of 50. mu.g/mL. After culturing the plate (right side up) in an incubator at 37 ℃ for 20-30min, the plate was inverted and cultured overnight.

Single colonies on plates were randomly picked and inoculated into 5mL LB medium containing 50. mu.g/mL Kan, cultured at 37 ℃ and 180rpm to OD₆₀₀When the value is about 0.8, 1mL of bacterial liquid is sent to a general biotechnology (Anhui) limited company for nucleotide sequence determination, 1mL of bacterial liquid is used for preserving strains, 3mL of bacterial liquid is used for extracting plasmids, and the specific steps refer to a plasmid small-amount extraction kit (Kangji century)) And (6) instructions. After the size and purity of a target band of a correctly sequenced strain plasmid are verified by 1% DNA agarose gel electrophoresis, 10mL of the plasmid is transferred into E.coli BL21(DE3) competent cells to obtain a target recombinant strain, and the rest of the plasmid is stored in a refrigerator at the temperature of 20 ℃ below zero for later use.

(4) Expression and purification of enzymes

Single colonies of the wild type and the mutant were picked and inoculated into 5mL LB liquid medium containing Kan at a final concentration of 50. mu.g/mL, and shake-cultured at 37 ℃ and 180rpm for 12 hours. The bacterial liquid is transferred to 200mL LB liquid culture medium containing 50 mu g/mL Kan with the final concentration by the inoculation amount (V/V) of 2 percent, and the culture is continued for 2-3 h under the conditions of 37 ℃ and 180 rpm. OD₆₀₀When the concentration reaches 0.6-0.8, IPTG with the final concentration of 0.5mM is added, and protein expression is induced under the conditions of 25 ℃ and 150 rpm. After inducing for 8-12 h, collecting the thallus at 6000r/min and 4 ℃.

The cells were washed 2 times with 0.02M PBS buffer (pH 7.8) to remove the residual medium and suspended in 30mL of a cell-breaking buffer (50mM sodium dihydrogenphosphate, 300mM sodium chloride, 20mM imidazole, pH 7.5). And carrying out ultrasonic disruption on the thalli cells under the ice bath condition (cell breaking condition: ultrasonic for 15min, working for 3s, intermittent for 6s and power 330 w). Centrifuging the cell disruption solution at 8000rpm and 4 deg.C for 45min, and collecting the supernatant as GAD-containing crude enzyme solution. And then, separating and purifying the crude enzyme solution by adopting a Ni-NTA affinity chromatography, loading, washing and eluting the crude enzyme to obtain a pure enzyme solution, and carrying out the operation steps according to the instruction.

(5) Determination of protein content

And (3) establishing a protein content standard curve by using a modified Bradford protein concentration determination kit, and determining the concentration of the pure enzyme, wherein the preparation steps of the protein standard curve are carried out according to the instruction. The molecular weight and purity of the purified protein were determined by SDS-PAGE (12% separation gel and 5% concentration gel).

The SDS-PAGE electrophoretograms of the wild type and the mutant are shown in FIG. 2. The electrophoresis bands of the wild type and the mutant are positioned on the same horizontal line, the molecular weight is about 54kDa and is consistent with the theoretical molecular weight, and a foundation is laid for the subsequent experiment.

(6) Determination of enzyme Activity

(1) Determination of enzyme Activity

mu.L of pure enzyme was reacted with 400. mu.L of a substrate solution (20mM sodium acetate buffer, 0.01mM PLP, 100mM L-Glu, pH 4.8) at 37 ℃ for 15min, and after completion of the reaction, 900. mu.L of NaHCO was added to 100. mu.L of the reaction product₃Solution (0.2M) to stop the reaction 500. mu.L of the above mixture and 500. mu.L of DNS-Cl acetone solution (4g/L) were subjected to derivatization reaction for 1 hour or more at 40 ℃ after derivatization, the GABA content was measured by HPLC, as described in (① Ueno Y HK, Takahashi S.purification and catalysis of methylation from Lacto bviruses IFO 12005.Biosci Biotech biochem 1997; 61 (637) 1168-71; ② Zhao AQ, HuXQ, Pan L, edition and catalysis of a biochemical reaction of amplification of a biochemical reaction and hydrolysis of Escherichia coli H3151. J.]Biotechnology yand Applied biochemistry.2018; 65(2):255-62.). The enzyme activity (U) is defined as the amount of enzyme required by GAD to catalyze the production of 1. mu. mol GABA from L-MSG per second under certain conditions.

The enzyme activities of the wild type and the 6 mutants are shown in figure 3, compared with the enzyme activity of the GAD wild type, the enzyme activity of the mutant D54A is obviously improved and is 1.55 times of that of the wild type, and the catalytic activity of the GAD is improved after the 54 site is replaced by the alanine. The enzyme activities of the other 5 mutants except the mutant D54A are reduced to different degrees, wherein the enzyme activity of the mutant E14R is 0.95 times of that of the wild type, and the enzyme activities of the mutant E14A, the mutant D54K, the mutant E114A and the mutant E114K are less than 60% of that of the wild type, which shows that the enzyme catalytic activity is reduced due to the amino acid substitution of the sites.

(2) Determination of residual Activity of enzymes

The purified wild type and mutant were incubated at 55 ℃ for 15min and immediately cooled on ice for 5min after incubation. Subsequently, 20. mu.L of the heat-treated enzyme solution was reacted with 400. mu.L of a substrate solution (20mM sodium acetate buffer, 0.01mM PLP, 100mM L-Glu, pH 4.8) at 37 ℃ for 15min, and the residual activities of the wild type and the mutant were determined. The experiments are carried out in parallel for three times, and the enzyme activity obtained without heat treatment is taken as 100 percent, so that the mutant with higher relative enzyme activity than the wild type is screened out.

The residual activity of the wild type and 6 mutants after heat treatment at 55 ℃ for 15min is shown in FIG. 4. The residual activities of mutant D54A, mutant D54K, mutant E114A and mutant E114K were all higher than that of the wild type, and were 1.24-fold, 1.23-fold, 1.09-fold and 1.08-fold, respectively, that of the wild type. The residual activities of mutant E14A and mutant E14R were lower than wild type, 71% and 53% of wild type residual activity, respectively.

In conclusion, by optimizing the surface charge of the protein, a mutant D54A with improved enzyme activity and thermostability was selected.

(7) Determination of enzymatic parameters

L-MSG substrate solutions were prepared at 5, 10, 20, 30, 40, 50, 70, 90, 100 concentrations with 0.01mM PLP in sodium acetate buffer (0.2M, pH 4.8). The enzyme activity of the GAD wild type and the mutant at different concentrations is determined by adopting an enzyme activity determination method. Different substrate concentrations [ S ]]The corresponding reaction rate V is brought into the Michaelis equation V ═ V_max×[S]/(K_m+[S]) Performing nonlinear fitting by using Origin 8.0 software, and calculating enzyme kinetic parameters K corresponding to wild type and mutant_mAnd V_max(ii) a By the formula k_cat＝V_max/[E]([E]Molar concentration of enzyme) to calculate k corresponding to wild type and mutant_catAnd k_cat/K_m。

As can be seen from Table 2, the affinity of mutant D54A for the substrate (K) is compared to the kinetic parameters of the wild type_m) Increase (K)_mLower values, stronger affinity of the enzyme for the substrate), k for mutant D54A_cat/K_mIs 1.80 times of the wild type. The above data indicate that mutant D54A increases its affinity for the substrate, increasing its catalytic efficiency for the substrate. Furthermore, compared to the wild type, the mutantsThe catalytic activity was significantly improved while the stability of variant D54A was improved.

TABLE 2 kinetic parameters of wild type and mutant

(8) Determination of thermal stability

(1)T₅₀ ¹⁵Measurement of (2)

T₅₀ ¹⁵The method is characterized in that the temperature corresponds to the temperature when the residual activity of the enzyme is reduced to 50% after the pure enzyme is incubated for 15min at the temperature of 30-70 ℃. Respectively incubating the purified wild enzyme and the mutant thereof at 30-70 ℃ for 15min, rapidly placing on ice for cooling for 5min after incubation is finished, and measuring the residual activity of the wild enzyme and the mutant thereof. Using temperature as abscissa, using ratio of enzyme activity after heat treatment and without heat treatment as ordinate, using Origin 8.0 software to make map, calculating T of wild type and mutant₅₀ ¹⁵。

(2)t_1/2Measurement of (2)

t_1/2The time corresponding to the time when the residual activity of the enzyme is reduced to 50% after the pure enzyme is incubated at 55 ℃ for different times is shown. And respectively incubating the purified wild type and the mutant thereof at 55 ℃ for 10-80 min, immediately cooling the wild type and the mutant thereof on ice for 5min after the incubation is finished, and measuring the residual activity of the wild type and the mutant thereof. Using time as abscissa, using ratio of enzyme activity after heat treatment to that without heat treatment as ordinate, using Origin 8.0 software to make map, and calculating t of wild type and mutant at 55 deg.C_1/2。

(3)T_mMeasurement of (2)

Wild-type and mutant T-cells were determined by Differential Scanning Fluorescence (DSF)_mThe measurement method was carried out by the conventional method (① Kim SJ, Lee JA, Joo JC, et. the same degree of thermal Stable CiP (Coprinus cinerea) through in silico design [ J ]].Biotechnology Progress.2010；26(4):1038-46.；②Niesen FH,Berglund H,VedadiM.The use of differential scanning fluorimetry to detect ligand interactionsthat promote protein stability[J]Nature protocols.2007; 2(9):2212-21.) are modified by mixing 1. mu.L 1 × SYPRO Orange dye with 49. mu.L 0.15mg/mL pure enzyme (pure enzyme diluted with buffer of 150mM NaCl and 50mM PBS, pH 7.8), placing in a quantitative fluorescence PCR instrument (version 2.2.2) to measure the change of fluorescence intensity of a sample at 28-56 ℃ with temperature rise, wherein the temperature rise rate is 0.7 ℃/s, each temperature lasts 30s, the excitation wavelength is 490nm, the emission wavelength is 605nm_m。

The results of the stability assay for mutant D54A are shown in fig. 5 and table 3. T of wild type₅₀ ¹⁵T of mutant D54A at 56.21 ℃₅₀ ¹⁵At 60.16 ℃ the D54A increased by 3.95 ℃ compared with the wild type. T of mutant D54A_1/2295.94min, is wild type t_1/214.76 times of (24.05 min). Furthermore, T of mutant D54A_mThe temperature is increased by 4.80 ℃ compared with the wild type (40.63 ℃). The above data indicate that mutant D54A has greater stability.

TABLE 3 stability parameters of wild type and mutant

Name (R)	T50 15(℃)	t1/2(min)	Tm(℃)
				Wild type	56.21±0.55	24.05±0.57	40.63±0.21
D54A	60.16±0.47	295.94+1.02	45.43+0.17

(9) Molecular dynamics simulation

Molecular Dynamics (MD) simulation is an effective method to analyze protein stability and its molecular mechanism. The Root Mean Square Deviation (RMSD) of the framework atoms indicates the stability of the whole protein, and is inversely related to the thermal stability of the protein. The root mean square fluctuations (RMS F) of individual residues represent the stability of individual amino acid residues in the protein structure, which is inversely related to the thermostability of the protein.

Taking the L.brevis CGMCC1306 GAD wild type three-dimensional structure as a template, and constructing the mutant three-dimensional structure by utilizing Pymol software. The wild type and mutant three-dimensional structures were treated with FoldX software (version 3.0beta5.1) and subjected to a molecular dynamics simulation of 10ns at 313K using Amber 14 force field of YASARA software (version 16.4.6). Loading three-dimensional structures of the wild type and the mutant in PDB format into YASARA software, and placing the structures after hydrotreating in water with the density of 0.998mg/L and the side length of 0

The sodium and chloride ions act as counter ions to make the system electrically neutral. The cutoff distance for van der Waals interaction is

The long-range electrostatic interaction was calculated using the Particle Mesh Ewald (PME) method. The time step is 2.5fs, the simulated three-dimensional structure conformation is stored once every 25ps, and the simulation track is visualized by utilizing Visual Molecular Dynamics (VMD) software.

Wild type and mutant D54A were each MD simulated at 313K for a duration of 10ns, the simulation results are shown in fig. 6. As can be seen from fig. 6a, the average RMSD of mutant D54A was significantly lower than the wild type throughout the MD simulation, indicating a higher stability of D54A. As can be seen from FIG. 6b, the RMSF of position 54 in the wild-type protein is higher than that of mutant D54A at the temperature of 313K, which indicates that the protein stability is improved after the alanine is replaced by aspartic acid at position 54 of GAD.

And (4) conclusion: the GAD mutant with higher catalytic performance is obtained by modifying the protein by optimizing the surface charge of the protein and combining the site-directed mutagenesis technology. K of mutant D54A compared to wild type_cat/K_mIs 1.80 times of wild type, t_1/214.76 times of wild type, T₅₀ ¹⁵And T_mThe temperature is increased by 3.95 ℃ and 4.80 ℃ respectively compared with the wild type.

Sequence listing

<110> Zhejiang science and technology institute

<120> glutamate decarboxylase mutant, gene and application

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<170>SIPOSequenceListing 1.0

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ctcaagtggg acttccgttt aaacaacgtg atttccatca atgcctccgg ccacaaatat 840

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ccccgtgaag ccgatcgatt ggttcgcgat caactattgg cggaaggaaa ctcgcggctg 180

aatctcgcca cgttctgtca gacttacatg gaaccggaag cggttgaact catgaaagat 240

acactggaga aaaacgccat cgataaatcc gagtatcctc ggaccgctga aattgaaaat 300

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ggcttggttt atcccggagt cggctgggta atctggcgtg accaacagta tctaccaaaa 900

gagctggtct ttaaggtcag ctacttgggt ggtgaactac ctacgatggc catcaacttc 960

tcccacagtg cctcccaatt aatcggtcag tattacaact ttattcgctt tggttttgat 1020

ggctatcgtg aaattcaaga aaaaactcac gacgttgccc gctatctcgc gaaatcgctc 1080

actaaattag ggggcttttc cctcattaat gacggccacg agttaccgct gatctgttat 1140

gaactcactg ccgattctga tcgcgaatgg accctctacg atttatccga tcggttatta 1200

atgaagggct ggcaggttcc cacctatccc ttaccaaaaa acatgacgga ccgcgttatt 1260

caacggatcg tggttcgggc tgactttggt atgagtatgg cccacgactt tattgatgat 1320

ctaacccaag ccattcacga tctcgaccaa gcacacatcg ttttccatag tgatccgcaa 1380

cctaaaaaat acggattcac tcactaa 1407

<210>3

<211>468

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>3

Met Ala Met Leu Tyr Gly Lys His Thr His Glu Thr Asp Glu Thr Leu

1 5 10 15

Lys Pro Ile Phe Gly Ala Ser Ala Glu Arg His Asp Leu Pro Lys Tyr

20 25 30

Lys Leu Ala Lys His Ala Leu Glu Pro Arg Glu Ala Asp Arg Leu Val

35 40 45

Arg Asp Gln Leu Leu Ala Glu Gly Asn Ser Arg Leu Asn Leu Ala Thr

50 55 60

Phe Cys Gln Thr Tyr Met Glu Pro Glu Ala Val Glu Leu Met Lys Asp

65 70 75 80

Thr Leu Glu Lys Asn Ala Ile Asp Lys Ser Glu Tyr Pro Arg Thr Ala

85 90 95

Glu Ile Glu Asn Arg Cys Val Asn Ile Ile Ala Asn Leu Trp His Ala

100 105 110

Pro Glu Ala Glu Ser Phe Thr Gly Thr Ser Thr Ile Gly Ser Ser Glu

115 120 125

Ala Cys Met Leu Ala Gly Leu Ala Met Lys Phe Ala Trp Arg Lys Arg

130 135 140

Ala Lys Ala Asn Gly Leu Asp Leu Thr Ala His Gln Pro Asn Ile Val

145 150 155 160

Ile Ser Ala Gly Tyr Gln Val Cys Trp Glu Lys Phe Cys Val Tyr Trp

165 170 175

Asp Ile Asp Met His Val Val Pro Met Asp Asp Asp His Met Ser Leu

180 185 190

Asn Val Asp His Val Leu Asp Tyr Val Asp Asp Tyr Thr Ile Gly Ile

195 200 205

Val Gly Ile Met Gly Ile Thr Tyr Thr Gly Gln Tyr Asp Asp Leu Ala

210 215 220

Arg Leu Asp Ala Val Val Glu Arg Tyr Asn Arg Thr Thr Lys Phe Pro

225 230 235 240

Val Tyr Ile His Val Asp Ala Ala Ser Gly Gly Phe Tyr Thr Pro Phe

245 250 255

Ile Glu Pro Glu Leu Lys Trp Asp Phe Arg Leu Asn Asn Val Ile Ser

260 265 270

Ile Asn Ala Ser Gly His Lys Tyr Gly Leu Val Tyr Pro Gly Val Gly

275 280 285

Trp Val Ile Trp Arg Asp Gln Gln Tyr Leu Pro Lys Glu Leu Val Phe

290 295 300

Lys Val Ser Tyr Leu Gly Gly Glu Leu Pro Thr Met Ala Ile Asn Phe

305 310 315 320

Ser His Ser Ala Ser Gln Leu Ile Gly Gln Tyr Tyr Asn Phe Ile Arg

325 330 335

Phe Gly Phe Asp Gly Tyr Arg Glu Ile Gln Glu Lys Thr His Asp Val

340 345 350

Ala Arg Tyr Leu Ala Lys Ser Leu Thr Lys Leu Gly Gly Phe Ser Leu

355 360 365

Ile Asn Asp Gly His Glu Leu Pro Leu Ile Cys Tyr Glu Leu Thr Ala

370 375 380

Asp Ser Asp Arg Glu Trp Thr Leu Tyr Asp Leu Ser Asp Arg Leu Leu

385 390 395 400

Met Lys Gly Trp Gln Val Pro Thr Tyr Pro Leu Pro Lys Asn Met Thr

405 410 415

Asp Arg Val Ile Gln Arg Ile Val Val Arg Ala Asp Phe Gly Met Ser

420 425 430

Met Ala His Asp Phe Ile Asp Asp Leu Thr Gln Ala Ile His Asp Leu

435 440 445

Asp Gln Ala His Ile Val Phe His Ser Asp Pro Gln Pro Lys Lys Tyr

450 455 460

Gly Phe Thr His

465

<210>4

<211>29

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

atgaaacaga tgcgacgctc aaaccaatc 29

<210>5

<211>29

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

tggtttgagc gtcgcatctg tttcatgcg 29

<210>6

<211>36

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

acgcatgaaa cagatcgtac gctcaaacca atcttc 36

<210>7

<211>36

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

gattggtttg agcgtacgat ctgtttcatg cgtgtg 36

<210>8

<211>28

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

tcaactattg gcggaaggaa actcgcgg 28

<210>9

<211>28

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

cgagtttcct tccgccaata gttgatcg 28

<210>10

<211>35

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

gcgatcaact attgaaagaa ggaaactcgc ggctg 35

<210>11

<211>35

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

ccgcgagttt ccttctttca atagttgatc gcgaa 35

<210>12

<211>32

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

agcggctgag tcgttcactg gcacctcgac ga 32

<210>13

<211>32

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

tgaacgactc agccgctgga gcatgccaga gg 32

<210>14

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>14

gcatgctcca aaagctgagt cgttc 25

<210>15

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>15

cgactcagct tttggagcat gccag 25

Claims (8)

1. A glutamic acid decarboxylase mutant, wherein the 54 th amino acid residue of wild-type glutamic acid decarboxylase is obtained by mutating aspartic acid to alanine.

2. The glutamate decarboxylase mutant according to claim 1, wherein the amino acid sequence is as shown in SEQ id No. 3.

3. A gene encoding the glutamate decarboxylase mutant according to claim 1 or 2.

4. The gene of claim 3, wherein the nucleotide sequence is shown in SEQ ID No. 2.

5. A genetically engineered bacterium comprising the gene of claim 4.

6. Use of a glutamate decarboxylase mutant according to claim 1 or 2 for catalyzing the removal of the α -carboxy group from L-glutamate for GABA production.

7. The use of the gene of claim 3 for catalyzing the production of GABA by removing alpha-carboxyl from L-glutamic acid.

8. The use of the gene of claim 4 for catalyzing the production of GABA by removing alpha-carboxyl from L-glutamic acid.

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