Preparation method of gamma-aminobutyric acid

Abstract

The invention relates to the technical field of biosynthesis, in particular to a preparation method of gamma-aminobutyric acid. The invention provides a method for preparing gamma-aminobutyric acid, according to the method, gamma-aminobutyric acid is catalytically synthesized by using a mutant of glutamate decarboxylase screened by the inventor, the problems of low catalytic efficiency, long conversion time and limited reuse times of enzyme are solved, the immobilized enzyme can be continuously subjected to at least 20 rounds of enzyme catalytic reaction, and the yield of gamma-aminobutyric acid is increased. And moreover, the catalytic efficiency is not obviously reduced, the substrate conversion rate can still be stabilized at 99% or above, and the method is suitable for industrial production.

Description

A method for preparing γ-aminobutyric acid Technical Field

[0001] This invention relates to the field of biosynthesis technology, specifically to a method for preparing γ-aminobutyric acid. Background Technology

[0002] Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter with various physiological functions, including lowering blood pressure, reducing anxiety, and improving sleep. It is widely used in the pharmaceutical, food, feed, and chemical industries. Currently, GABA is prepared through chemical synthesis, plant extraction, and microbial fermentation, but these methods suffer from problems such as high cost, low yield, and complex processes.

[0003] While there are existing reports of using glutamate decarboxylase to catalyze the production of GABA from L-glutamate, the poor thermal stability, low catalytic efficiency, and limited number of reusable enzymes restrict their industrial application. Therefore, developing an efficient, stable, and reusable GABA preparation process has significant industrial value. Summary of the Invention

[0004] This invention aims to at least partially solve one of the technical problems in related technologies. Therefore, one objective of this invention is to provide a method for preparing γ-aminobutyric acid (GABA). This method utilizes a mutant of glutamate decarboxylase screened by the inventors to catalyze the synthesis of GABA, solving the problems of low enzyme catalytic efficiency and limited reusability. The immobilized enzyme can continuously perform at least 20 rounds of enzyme catalytic reactions without significant reduction in catalytic efficiency, and the substrate conversion rate remains stable at over 99%, making it suitable for industrial production.

[0005] Therefore, a first aspect of the present invention provides a method for preparing γ-aminobutyric acid (GABA). According to an embodiment of the present invention, the method comprises: adding a mutant of glutamate decarboxylase or wet bacterial cells containing said glutamate decarboxylase to a reaction system containing L-glutamate and pyridoxal phosphate, and catalytically synthesizing γ-aminobutyric acid, wherein the amino acid sequence of said glutamate decarboxylase mutant is obtained by mutating at least one of the following positions of the amino acid sequence of wild-type glutamate decarboxylase: positions 278, 282, 285, and 405, wherein the amino acid sequence of said wild-type glutamate decarboxylase is as shown in SEQ ID NO: 1.

[0006] 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.

[0007] By utilizing the mutant of this glutamate decarboxylase, γ-aminobutyric acid was synthesized through catalysis, which solved the problems of low catalytic efficiency and limited number of reuses of the enzyme. The immobilized enzyme can continuously carry out at least 20 rounds of enzyme catalytic reaction without significant reduction in catalytic efficiency, and the substrate conversion rate can still remain stable at over 99%, making it suitable for industrial production.

[0008] According to an embodiment of the present invention, compared with the amino acid sequence of the 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) isoleucine at position 285 is mutated to valine or leucine; (4) proline at position 405 is mutated to threonine.

[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 the following mutations 1)-10): 1) Tyrosine at position 278 is mutated to phenylalanine; or 2) Tyrosine at position 282 is mutated to phenylalanine; or 3) Isoleucine at position 285 is mutated to valine; or 4) Isoleucine at position 285 is mutated to leucine; or 5) Proline at position 405 is mutated to threonine; or 6) Tyrosine at position 278 is mutated to phenylalanine. 7) Isoleucine at position 285 is mutated to valine; or 8) Tyrosine at position 282 is mutated to phenylalanine, and isoleucine at position 285 is mutated to valine; 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.

[0010] According to an embodiment of the present invention, the mutant of glutamate decarboxylase added to the reaction system is derived from wet cells obtained by fermentation culture.

[0011] According to an embodiment of the present invention, the mutant of glutamate decarboxylase added to the reaction system is derived from the crude enzyme solution obtained by crushing and centrifuging the wet bacterial cells.

[0012] According to an embodiment of the present invention, the weight ratio of the wet bacterial cells to the substrate L-glutamic acid is 1:(20~30).

[0013] According to an embodiment of the present invention, the method further includes: purifying and / or immobilizing the crude enzyme solution before the enzyme-catalyzed reaction occurs.

[0014] According to an embodiment of the present invention, the purified crude enzyme solution is immobilized using an enzyme immobilization carrier resin.

[0015] According to an embodiment of the present invention, the enzyme immobilization carrier resin includes those selected from LX-109S and ES-103B.

[0016] According to an embodiment of the present invention, the reaction temperature for the catalytic synthesis of γ-aminobutyric acid is 50-70 °C, and the reaction time is at least 2 h.

[0017] This invention provides a glutamate decarboxylase mutant with higher catalytic efficiency to address 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 or a combination thereof, using the amino acid sequence shown in SEQ ID NO: 1 as the starting sequence: tyrosine at position 278 mutated to phenylalanine, tyrosine at position 282 mutated to phenylalanine, isoleucine at position 285 mutated to valine or leucine, and proline at position 405 mutated to threonine; and the mutant possesses glutamate decarboxylase activity. Using the glutamate decarboxylase mutant provided by this invention, the problems of low catalytic efficiency, long conversion time, and limited number of reuses in the preparation of γ-aminobutyric acid (GABA) are solved. The immobilized enzyme can continuously perform at least 20 rounds of enzyme catalytic reaction without significant reduction in catalytic efficiency, and the substrate conversion rate remains stable at over 99%, meeting the industrial demand for efficient and low-cost preparation of GABA.

[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 shows the three-dimensional spatial structure of the protein obtained by protein modeling of FcGAD using software tools; Figure 2 shows the SDS-PAGE gel electrophoresis results of the supernatant and precipitate obtained after culturing mutant B1-B12 genetically engineered bacteria; Figure 3 shows the SDS-PAGE gel electrophoresis results of the supernatant and precipitate obtained after culturing double mutant B13-B16 genetically engineered bacteria, with B5 genetically engineered bacteria as a control; Figure 4 shows the SDS-PAGE gel electrophoresis results of the supernatant and precipitate obtained after culturing triple mutant B17 genetically engineered bacteria, with B15 genetically engineered bacteria as a control; Figure 5 shows the SDS-PAGE gel electrophoresis results of crude enzyme solutions of FcGAD and FcGAD-B17. 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 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 method for preparing γ-aminobutyric acid (GABA), comprising: adding a mutant of glutamate decarboxylase or wet bacterial cells containing said glutamate decarboxylase to a reaction system containing L-glutamate and pyridoxal phosphate, and catalytically synthesizing GABA, wherein the amino acid sequence of said glutamate decarboxylase mutant is obtained by mutating at least one of the following positions of the amino acid sequence of wild-type glutamate decarboxylase: position 278, position 282, position 285, and position 405, wherein the amino acid sequence of said wild-type glutamate decarboxylase is as shown in SEQ ID NO: 1.

[0027] SEQ ID NO: 1 looks like this: MLYGKKNREEEEYLTPIFGSSAEGEDLPKYKLNKESIEPRIAYRLVKDQLLDEGNARLNLATFCQTYMEPEATKLMAETLEKNAIDKSEYPRTAEIENRCVNIIADLWHAPKDEKFLGTSTVGSSEACLMLGGMAMKFAWRNRAEKLGLDI NAKKPNLVISSGYQVCWEKFCVYWDIEMRTVPMDEDHMSLNVDKVLDYVDEYTIGIVGILGITYTGKYDDIKALDDLVEKYNQTTDYKVYIHVDAASGGFFTPFVEPELEWDFRLKNVISINTSGHKYGLVYPGIGWVLWRDQEYLPKELIFKVSY According to a specific embodiment of the present invention, compared with the amino acid sequence of the 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) isoleucine at position 285 is mutated to 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 mutations 1)-10): 1) Tyrosine at position 278 is mutated to phenylalanine; or 2) Tyrosine at position 282 is mutated to phenylalanine; or 3) Isoleucine at position 285 is mutated to valine; or 4) Isoleucine at position 285 is mutated to leucine; or 5) Proline at position 405 is mutated to threonine; or 6) Tyrosine at position 278 is mutated to phenylalanine. The following are examples of amino acids: 1) Isoleucine at position 285 is mutated to valine; 2) Tyrosine at position 282 is mutated to phenylalanine, and isoleucine at position 285 is mutated to valine; 3) Isoleucine at position 285 is mutated to valine, and proline at position 405 is mutated to threonine; 4) Tyrosine at position 278 is mutated to phenylalanine, and isoleucine at position 285 is mutated to leucine; 5) 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 mutant of glutamate decarboxylase added to the reaction system is derived from wet cells obtained by fermentation culture.

[0031] According to a specific embodiment of the present invention, the mutant of glutamate decarboxylase added to the reaction system is derived from the crude enzyme solution obtained by crushing and centrifuging the wet bacterial cells.

[0032] It should be noted that there are no particular restrictions on the conditions for disruption and centrifugation. For example, cell lysate can be obtained by ultrasonic or high-pressure homogenization. There are also no particular restrictions on the centrifugation parameters, which can be within the range of parameters commonly used in this field, as long as the enzyme is separated from the wet bacterial cells.

[0033] According to a specific embodiment of the present invention, the weight ratio of the wet bacterial cells to the substrate L-glutamic acid is 1:(20~30).

[0034] According to a specific embodiment of the present invention, the method further includes: purifying and / or immobilizing the crude enzyme solution before the enzyme-catalyzed reaction occurs.

[0035] According to a specific embodiment of the present invention, the purified crude enzyme solution is immobilized using an enzyme immobilization carrier resin.

[0036] According to a specific embodiment of the present invention, the enzyme immobilization carrier resin includes, but is not limited to, LX-109S, ES-103B, etc. It should be noted that even for the same bacterial cell, different enzyme immobilization carrier resins may have slightly different immobilization efficiencies, which is understandable to those skilled in the art.

[0037] According to a specific embodiment of the present invention, the reaction temperature for the catalytic synthesis of γ-aminobutyric acid is 50-70 °C, and the reaction time is at least 2 hours. For example, the reaction temperature for the catalytic synthesis of γ-aminobutyric acid is 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, etc., and the reaction time is 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, etc. Preferably, the reaction temperature for the catalytic synthesis of γ-aminobutyric acid is 55-60 °C, and the reaction time is 2-4 hours.

[0038] According to a specific embodiment of the present invention, the present invention provides a method for preparing γ-aminobutyric acid, specifically including: (1) preparation of crude enzyme solution of glutamate decarboxylase: the prepared glutamate decarboxylase mutant is inoculated into a fermentation medium for fermentation culture, and the wet cells of the mutant are obtained from the fermentation product. The wet cells are crushed and centrifuged to obtain crude enzyme solution; (2) purification and immobilization of crude enzyme solution; (3) reaction under certain conditions to obtain γ-aminobutyric acid; (4) extraction and collection of γ-aminobutyric acid.

[0039] According to a specific embodiment of the present invention, the method for preparing γ-aminobutyric acid (GABA) further includes purifying the reaction solution obtained from the enzyme-catalyzed reaction to obtain pure GABA. It should be noted that there are no particular limitations on the purification method. For example, adsorption and decolorization can be performed using a resin column, followed by filtration. Furthermore, the filtrate can be subjected to vacuum distillation, cooling crystallization, and centrifugation to obtain pure GABA. Of course, other purification methods can also be used, all of which are within the scope of protection of the present invention.

[0040] 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.

[0041] 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 recovery and 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 of the plasmid extraction kit; the DNA gel recovery procedure followed the instructions of the DNA gel recovery and purification kit; the DNA fragment ligation procedure followed the instructions of the one-step rapid cloning kit; the BCA protein detection kit procedure followed the instructions of the BCA protein detection kit. 2) LB medium (g / L): peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, sterilized at 121℃ for 20 min.

[0042] 3) Fermentation medium (g / L): peptone 10 g / L, yeast powder 5 g / L, glycerol 10 g / L, disodium hydrogen phosphate dodecahydrate 20 g / L, potassium dihydrogen phosphate 7 g / L, ammonium chloride 5 g / L, anhydrous sodium sulfate 1 g / L and magnesium sulfate heptahydrate 1 g / L, defoamer 0.5 ml / L, sterilized at 121℃ for 30 min.

[0043] 4) Enzyme activity determination: 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 bacteria 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 ODS-2 Hypersil™ (4.6 × 250 mm, 5 µm); flow rate 1.0 mL / min; detection wavelength 210 nm; mobile phase: methanol: 0.05 mol / L potassium dihydrogen phosphate + 0.005 mol / L sodium decane sulfonate (pH = 2.30) = 7:93.

[0044] 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.

[0045] Example 1: Construction of Wild-Type Glutamic Acid Decarboxylase FcGAD Genetically Engineered Bacteria (1) Synthesis of Wild-Type FcGAD Gene The amino acid sequence of glutamate decarboxylase FcGAD derived from Lactobacillus brevis is shown in SEQ ID NO: 1. Based on the codon preference rules of Escherichia coli, the gene coding sequence was codon optimized. After adding NdeI and BamHI restriction sites at both ends of the coding sequence, it was sent to a biotechnology company for artificial synthesis to obtain FcGAD (SEQ ID NO: 2).

[0046]

[0047] (3) Construction of wild-type FcGAD genetically engineered bacteria: The homologous recombination product obtained in (2) was transformed into Escherichia coli BL21 (DE3) competent cells by heat shock method. The transformation product was plated on LB plates containing 50 μg / mL ampicillin and cultured overnight at 37°C. Positive transformants were selected and sent to a gene company for sequencing. Transformants with correct sequencing contained the wild-type FcGAD gene plasmid vector FcGAD-pET15b. This transformant was also a wild-type FcGAD genetically engineered bacterium.

[0048] 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 structural quality indicators such as sequence consistency, coverage, template resolution, and R-factor, CGMCC 1306 (PDB ID: 5GP4) from Lactobacillus brevis was ultimately selected as the modeling template. PyMol was used to visualize and process the obtained model. The model was evaluated through stereochemical quality analysis (Ramachandran diagram analysis) and three-dimensional profile-sequence compatibility analysis. The results are shown in Figure 1. 97% of the residues are located within allowed regions, and only 0.2% of the residues (located in highly flexible loop regions) are located within disallowed regions, indicating excellent skeletal characterization. Based on literature reports, FcGAD has a dimer structure and a trimer structure in crystals (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 bonds 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. The results showed that the substrate interacts with residues such as R420, H276, and K277. Therefore, to improve the enzyme activity of FcGAD, we mainly screened the substrate, PLP, and residues within 5 Å around the loop (Y306-E312). We also predicted the free energy change after mutation using the ProStab online website. A negative ΔΔG value indicates that the mutation makes the protein more stable, which is beneficial for improving activity. The mutation sites are shown in Table 1 below.

[0049] Table 1:

[0050] Example 3: Preparation and expression of FcGAD single mutant. The original plasmid was used as a template for site-directed mutagenesis to obtain the recombinant plasmid FcGAD-X-pET15b carrying the glutamate decarboxylase mutant. The site-directed mutagenesis method is as follows: The primer sequences used to obtain the mutant are shown in Table 2 (the bolded part in the primer sequence is the base corresponding to the mutation site).

[0051] Table 2:

[0052] 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.

[0053] 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.

[0054] 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. 600 The concentration was 0.6-0.8, and IPTG was added to a final concentration of 0.2 mM. The mixture was cultured at 20℃ and 200 rpm for 18 h. After centrifuging the fermentation broth 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 bacterial cell concentration of 100 mg / mL, sonicated for 10 min, centrifuged to remove the pellet, and the supernatant was collected for SDS-PAGE gel electrophoresis analysis. The results are shown in Figure 2, indicating that these mutant proteins were all expressed normally.

[0055] Example 4: FcGAD Single Mutant Enzyme Activity Assay 25 μL of enzyme solution was added to a centrifuge tube containing 975 μL of substrate solution (water containing 200 mM L-glutamic acid and 0.1 mM pyridoxal phosphate (PLP)). The mixture was incubated at 50°C for 30 min. After the reaction was complete, an equal volume of 10% hydrochloric acid solution was added to terminate the reaction. The mixture was shaken well and centrifuged. The supernatant was then analyzed by HPLC, using L-glutamic acid and γ-aminobutyric acid standards as controls.

[0056] The relative enzyme activities of each single mutant are shown in Table 3.

[0057] Table 3:

[0058] 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.

[0059] Example 5: Preparation and Expression of FcGAD Double Mutants. Five mutants were selected from mutation sites with significantly increased enzyme activity (approximately 20% or more) compared to the wild-type enzyme, namely B2, B3, B5, B6, and B9 in Table 3. The base sequences of the corresponding genes were analyzed, and amino acid mutation information was analyzed. Combination mutations were performed and 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 in Figure 3, indicating that these double mutant proteins were expressed normally. The protein expression and relative enzyme activity of the obtained double mutants were measured using the same method as in Example 4, and the results are shown in Table 4.

[0060] Table 4:

[0061] 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.

[0062] Example 6: Preparation and Expression of FcGAD Triple Mutants. Three double mutants with significantly increased enzyme activity prepared in Example 5, namely B13, B15, and B16, were selected. The base sequences of the corresponding genes were analyzed, and amino acid mutation information was examined. Combined mutations were 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.

[0063] Table 5:

[0064] 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.

[0065] Example 7: Fermentation culture of bacterial strains. Take 1 mL each of the genetically engineered strain FcGAD (producing wild-type glutamate decarboxylase) and the genetically engineered strain FcGAD-B17 (a mutant of glutamate decarboxylase) prepared in Example 1 and Example 6, activate them on LB slant medium, and culture at 37℃ for 16 h; pick an inoculation loop of bacterial growth from the slant medium and transfer it to LB seed medium containing 100 mL, and culture at 37℃ and 160 rpm with shaking for 10-16 h. Inoculate the seeds at a volume ratio of 8% into a 5 L fermenter containing 3 L of fermentation medium. Control the temperature at 37℃±1℃, maintain the pH at 7.0±0.1 during fermentation, control the aeration rate at 0.5 vvm~3.0 vvm, and control the rotation speed at 400 rpm~600 rpm. Maintain dissolved oxygen at 30% by adjusting the rotation speed and aeration rate. After fermentation for 7~8 h, cool down and stabilize to 20~30℃, add IPTG at a final concentration of 0.2 mM to induce fermentation for about 18 h. Stop fermentation when the OD of fermentation no longer increases, and obtain the fermentation broth.

[0066] Centrifuge the fermentation broth at 6000 rpm for 20 min, discard the supernatant to obtain wet cells. Resuspend the cells in water to a final concentration of 100 g / L, then homogenize the bacterial solution under high pressure to obtain cell lysate. Centrifuge the lysate supernatant to obtain the crude enzyme solution.

[0067] The crude enzyme solutions of FcGAD and FcGAD-B17 were analyzed by SDS-PAGE gel electrophoresis, and the results are shown in Figure 5. The results indicate that both FcGAD and FcGAD-B17 proteins are expressed normally.

[0068] Example 8: Purification of glutamate decarboxylase protein. 100 uM PLP was added to the crude enzyme solutions of FcGAD and FcGAD-B17 obtained in Example 7 and incubated at room temperature for 1 h. After incubation, the solutions were placed in a 65℃ water bath for 60 min, and then centrifuged at 6000 rpm for 15 min. The supernatant was then collected, which was the purified glutamate decarboxylase solution.

[0069] Example 9: Immobilization of glutamate decarboxylase protein. A suitable enzyme immobilization carrier resin was selected for immobilization. The specific steps of enzyme immobilization are as follows: (1) Resin equilibration: 875g (wet weight) of resin was weighed into a container, and 3 times its weight of 2 M pH=7.5 K2HPO4-KH2PO4 buffer solution was added. The container was allowed to stand and soak for 2 hours. During this period, the container could be stirred appropriately to ensure more thorough contact. After soaking, the container was filtered, the resin was collected, and the used buffer solution was discarded. The collected resin was put back into the container, and the soaking and filtration process was repeated. This cycle was repeated a total of 3 times.

[0070] (2) Immobilization: 17.5 L of the purified glutamate decarboxylase solution obtained in Example 8 was transferred to a container of resin treated in (1), and an equal volume of the phosphate buffer solution from (1) was added. The mixture was gently stirred at 27 °C for 16 h. (3) Filtration and washing: After immobilization, the resin was filtered, and the filtrate was retained as a sample. The resin was washed with 4 times its mass of 100 μm PLP solution and filtered, repeated 2-4 times. The washing solution was collected, and the concentration of unimmobilized enzyme in the filtrate and washing solution was detected to evaluate the resin immobilization efficiency.

[0071] The results showed that the immobilization efficiencies of enzyme immobilization carrier resins LX-109S and ES-103B (both purchased from Xi'an Lanxiao Technology New Material Co., Ltd.) for FcGAD-B17 protein were 87.6% and 83.7%, respectively; and the immobilization efficiencies of LX-109S and ES-103B for FcGAD protein were 84.3% and 79.2%, respectively.

[0072] Example 10: Enzymatic Conversion for the Production of γ-Aminobutyric Acid. FcGAD and FcGAD-B17 immobilized with the highly efficient LX-109S carrier resin were placed in stainless steel reaction vessels. 1.72 g PLP and 65 kg RO water were added, and the temperature was raised to 55-60°C. 44 kg of L-glutamic acid was then added for the reaction. During the reaction, when there was no undissolved solid matter other than the enzyme immobilization carrier resin in the vessel and no bubbles were generated, a sample was taken to measure the L-glutamic acid concentration. When the L-glutamic acid concentration was ≤2 g / L, the reaction endpoint was reached, and the reaction time from start to finish was recorded. The post-reaction solution was transferred to a transfer tank, while the enzyme immobilization carrier resin remained in the reaction vessel. The above steps were repeated for multiple rounds of reaction. The reaction conditions of FcGAD and FcGAD-B17 enzyme immobilization carrier resins are shown in Tables 6 and 7.

[0073] Table 6: Reaction status of FcGAD enzyme immobilization carrier resin

[0074] Table 7: Reaction status of FcGAD-B17 enzyme immobilization carrier resin

[0075] The results showed that the FcGAD enzyme immobilization carrier resin underwent a total of 16 rounds of reaction. Starting from the 12th round, the conversion rate decreased significantly. By the 16th round, the concentration of unreacted L-glutamate reached 57 g / L, and the conversion rate was only 86%. In contrast, the FcGAD-B17 enzyme immobilization carrier resin could continuously undergo at least 20 rounds of enzyme catalytic reaction without a significant decrease in catalytic efficiency. The substrate conversion rate remained stable at over 99%, with an average reaction time of 3.1 h and a final average L-glutamate concentration of 0.33 g / L.

[0076] Example 11: Purification of γ-aminobutyric acid. Any reaction solution obtained in Example 10 was pumped into a resin column for adsorption and decolorization. The decolorized solution was filtered through a 0.2 μm filter. The filtrate was subjected to vacuum distillation, cooling crystallization, and further centrifugation to obtain wet γ-aminobutyric acid. After drying, pure γ-aminobutyric acid was obtained. The pure product was tested, and the results are shown in Table 8: Table 8:

[0077] The results show that the γ-aminobutyric acid obtained by this post-extraction treatment has a high purity, with an average purity of 99.6%.

[0078] 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.

[0079] 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 method for preparing γ-aminobutyric acid, characterized in that, include: A mutant of glutamate decarboxylase or wet bacterial cells containing the mutant of said glutamate decarboxylase are added to a reaction system containing L-glutamate and pyridoxal phosphate to catalyze the synthesis of γ-aminobutyric acid (GABA), wherein the amino acid sequence of said glutamate decarboxylase mutant is obtained by mutating at least one of the following positions of the amino acid sequence of wild-type glutamate decarboxylase: positions 278, 282, 285, and 405, wherein the amino acid sequence of said wild-type glutamate decarboxylase is as shown in SEQ ID NO:

1.

2. The method 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) isoleucine at position 285 is mutated to valine or leucine; (4) proline at position 405 is mutated to threonine.

3. The method 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 mutations 1)-10): 1) Tyrosine at position 278 is mutated to phenylalanine; or 2) Tyrosine at position 282 is mutated to phenylalanine; or 3) Isoleucine at position 285 is mutated to valine; or 4) Isoleucine at position 285 is mutated to leucine; or 5) 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 leucine. 7) Isoleucine at position 282 is mutated to phenylalanine, and isoleucine at position 285 is mutated to valine; or 8) Isoleucine at position 285 is mutated to valine, and proline at position 405 is mutated to threonine; or 9) Isoleucine at position 278 is mutated to phenylalanine, and isoleucine at position 285 is mutated to leucine; or 10) Isoleucine 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. The method according to claim 1, characterized in that, The mutant glutamate decarboxylase added to the reaction system was derived from wet cells obtained through fermentation culture.

5. The method according to claim 4, characterized in that, The mutant glutamate decarboxylase added to the reaction system was derived from the crude enzyme solution obtained after the wet bacterial cells were crushed and centrifuged.

6. The method according to claim 4, characterized in that, The weight ratio of the wet bacterial cells to the substrate L-glutamic acid is 1:(20~30).

7. The method according to claim 5, characterized in that, The method further includes: purifying and / or immobilizing the crude enzyme solution before the enzyme-catalyzed reaction occurs.

8. The method according to claim 7, characterized in that, The purified crude enzyme solution was immobilized using an enzyme immobilization carrier resin.

9. The method according to claim 8, characterized in that, The enzyme immobilization carrier resin includes those selected from LX-109S and ES-103B.

10. The method according to claim 1, characterized in that, The reaction temperature for the catalytic synthesis of γ-aminobutyric acid is 50-70 °C, and the reaction time is at least 2 h.

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