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

Abstract

The invention belongs to the technical field of enzyme engineering, and particularly relates to a glutamate decarboxylase PmGAD mutant and application thereof in synthesis of gamma-aminobutyric acid. The mutant is obtained by mutating at least one amino acid in the 465 Phe, the 276 Ser, the 249 Ser, the 130 Glu, the 66 Thr, the 213 Gly, the 65 Ala and the 215 Thr on the basis of SEQ ID NO: 1. Compared with wild-type glutamate decarboxylase, the glutamate decarboxylase PmGAD mutant disclosed by the invention has relatively high catalytic efficiency, and can be used for efficiently synthesizing gamma-aminobutyric acid through a whole-cell catalytic reaction in only 8 hours, and the conversion rate reaches 99% or above.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to a glutamate decarboxylase PmGAD mutant and its application in the synthesis of γ-aminobutyric acid. Background Technology

[0002] Gamma-aminobutyric acid (GABA), as the most important inhibitory neurotransmitter in the mammalian central nervous system, has a variety of significant physiological functions, including lowering blood pressure, reducing anxiety, improving sleep, promoting growth hormone secretion, and regulating immune function. In recent years, with the increasing demand for health-functional foods and drugs, the application value of GABA in food, medicine, cosmetics, and feed additives has become increasingly prominent.

[0003] Currently, the main methods for industrial-scale GABA production include chemical synthesis, plant enrichment, and microbial synthesis. Among these, microbial synthesis has become the most promising GABA production method due to its environmental friendliness, high safety, and low production cost. In the microbial synthesis of GABA, glutamate decarboxylase (GAD, EC4.1.1.15) is the key catalytic enzyme. With the participation of the cofactor pyridoxal phosphate (PLP), this enzyme specifically removes the α-carboxyl group from the L-glutamate molecule through an α-decarboxylation reaction, ultimately producing GABA and carbon dioxide. This catalytic reaction is highly specific and irreversible, making it the core reaction step in the microbial synthesis of GABA. However, the wild-type GABA currently used in industrial applications has a significant drawback: low enzyme activity, which severely restricts the production efficiency and industrialization process of GABA. Summary of the Invention

[0004] The purpose of this invention is to provide a PmGAD mutant of glutamate decarboxylase and its application in the synthesis of γ-aminobutyric acid, so as to solve the problems of low enzyme activity and low catalytic efficiency of wild-type glutamate decarboxylase.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a glutamate decarboxylase PmGAD mutant, which is obtained by mutating at least one amino acid from the following positions: Phe at position 465, Ser at position 276, Ser at position 249, Glu at position 130, Thr at position 66, Gly at position 213, Ala at position 65, and Thr at position 215, based on SEQ ID NO:1.

[0006] Preferably, the mutant is based on SEQ ID NO:1, with the Phe at position 465 mutated to Leu; or Mutate the 276th Ser bit to Pro; or Mutate the 249th Ser position to Phe; or Mutate Glu at position 130 to His; or Mutate the 66th Thr to Met; or Mutate Gly at position 213 to Arg; or Mutate the 66th Thr to Asp; or Mutate the 65th Ala to Ala (synonymous mutation); or Mutate Gly at position 213 to Arg; or Mutate Thr at position 215 to Phe; or Mutate the 215th Thr to Thr (synonymous mutation); or The Phe at position 465 was mutated to Ile.

[0007] More preferably, the mutant is based on SEQ ID NO:1, with Phe at position 465 mutated to Leu and Ser at position 276 mutated to Pro (its amino acid sequence is shown in SEQ ID NO:3, and its nucleotide sequence is shown in SEQ ID NO:4); or Mutate Phe at position 465 to Leu and mutate Ser at position 249 to Phe (the amino acid sequence is shown in SEQ ID NO:5, and the nucleotide sequence is shown in SEQ ID NO:6); or The 276th Ser was mutated to Pro, and the 249th Ser was mutated to Phe (the amino acid sequence is shown in SEQ ID NO:7, and the nucleotide sequence is shown in SEQ ID NO:8).

[0008] More preferably, the mutant is based on SEQ ID NO:1, with Phe at position 465 mutated to Leu, Ser at position 276 mutated to Pro, and Ser at position 249 mutated to Phe (its amino acid sequence is shown in SEQ ID NO:9, and its nucleotide sequence is shown in SEQ ID NO:10).

[0009] The present invention also provides a gene encoding any of the above-mentioned glutamate decarboxylase PmGAD mutants and an expression vector carrying the gene.

[0010] Furthermore, the recombinant expression vector uses pET-21b(+) as the original expression vector.

[0011] The present invention also provides recombinant strains comprising the expression vector.

[0012] Preferably, the host bacterium of the recombinant bacteria is Escherichia coli. Escherichia coli BL21(DE3).

[0013] Application of any of the above-mentioned glutamate decarboxylase PmGAD mutants in the production of γ-aminobutyric acid.

[0014] A method for producing γ-aminobutyric acid includes the following steps: Add any of the above-mentioned glutamate decarboxylase PmGAD mutants or the recombinant strains to a reaction system containing L-glutamate and pyridoxal 5-phosphate (PLP), and carry out the decarboxylation reaction at pH 3.0-6.0 and 30-50℃ to generate γ-aminobutyric acid.

[0015] Preferably, the concentration of L-glutamic acid in the reaction system is 100-600 g / L, and the concentration of the recombinant strain is 10-100 g / L.

[0016] The beneficial effects of this invention are as follows: This invention utilizes directed evolution technology to study bacteria derived from Bacillus megaterium. Priestia megaterium The wild-type glutamate decarboxylase PmGAD (amino acid sequence shown in SEQ ID NO:1, nucleotide sequence shown in SEQ ID NO:2) (NCBI protein sequence number ALU66010.1) was modified, resulting in a glutamate decarboxylase mutant with an enzyme activity 2.58 times higher than the wild-type glutamate decarboxylase. Using this mutant for whole-cell catalytic synthesis of γ-aminobutyric acid (GABA) offers advantages such as high conversion efficiency (over 99% conversion rate in just 8 hours) and mild reaction conditions. This invention provides a new technical approach for the green and efficient synthesis of GABA. Attached Figure Description

[0017] Figure 1 Image of the expression vector for glutamate decarboxylase PmGAD.

[0018] Figure 2 The liquid chromatograms of L-glutamic acid and GABA during whole-cell catalysis using L-glutamic acid as a substrate are shown.

[0019] Figure 3 The reaction equation is catalyzed by glutamate decarboxylase. Detailed Implementation

[0020] The following examples are provided to further illustrate the present invention, but do not limit the invention in any way. Processes and methods not described in detail in the following examples are conventional methods known in the art, and the reagents used in the examples are commercially available or prepared by methods well known to those skilled in the art. The following examples all achieve the objectives of the present invention.

[0021] The culture media involved in the following examples are as follows: LB liquid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, sterilized at 121°C for 20 min; LB solid medium: LB liquid medium with 2% agar added; TB liquid culture medium: KH2PO4 2.31 g / L, K2HPO4·3H2O 16.42 g / L, yeast extract 24 g / L, peptone 12 g / L, glycerol 4 g / L.

[0022] Example 1: Preparation of genetically engineered strains containing the glutamate decarboxylase PmGAD gene The glutamate decarboxylase mutant described in this invention is derived from Bacillus megaterium using directed evolution technology. Priestia megaterium The wild-type glutamate decarboxylase (NCBI protein sequence number ALU66010.1) was modified by modifying its amino acid sequence. The amino acid sequence of the wild-type glutamate decarboxylase is shown in SEQ ID NO:1, with a total length of 467 amino acids, and the nucleotide sequence is shown in SEQ ID NO:2, with a total length of 1404 bases.

[0023] After designing primers, the gene for wild-type glutamate decarboxylase from Bacillus megaterium was cloned into the pET21b vector using homologous recombination, constructing the recombinant vector pET21b-PmGAD. This recombinant vector was then transformed into E. coli BL21(DE3) competent cells and plated on LB agar plates containing ampicillin sodium (Amp), incubated upside down for 12-16 hours. Single colonies were picked for colony PCR identification; correctly identified positive transformants were the genetically engineered strains containing the glutamate decarboxylase PmGAD gene.

[0024] Example 2: Construction of a point mutation library of the glutamate decarboxylase PmGAD gene (1) Based on the spatial structure of glutamate decarboxylase PmGAD and its spatial position in binding with substrates and cofactors, 23 amino acid sites (A65, T66, F67, V68, G127, S128, S129, E130, C132, Q166, V168, W169, I211, G213, T215, D246, A248, S249, S276, H278, K279, R422, F465) were selected for saturation mutagenesis.

[0025] (2) Construct engineered strains expressing 23 PmGAD mutant glutamate decarboxylase genes. The PmGAD mutant glutamate decarboxylase was obtained by designing degenerate primers to construct a mutant library for screening. Four forward mutagenesis primers were designed using the Tang method to uniformly and without redundancy introduce twenty natural amino acids into the mutant library. The designed degenerate primer sequences are shown below, and the specific construction method is as follows: Using the recombinant vector pET21b-PmGAD constructed in Example 1 as a template, two rounds of PCR reactions were performed using primers corresponding to each mutant. The PCR reaction system for the first round is shown in Table 1, the PCR reaction system for the second round is shown in Table 2, and the PCR reaction procedure is shown in Table 3.

[0026] Primer names and sequences for constructing the point mutation library of the glutamate decarboxylase PmGAD gene unit: A65 mutation library: GAD-A65-NDT-F1: GCCCGCTTGAATTTANDTACGTTTGTTACTACG; GAD-A65-VMA-F2: GCCCGCTTGAATTTAVMAACGTTTGTTACTACG; GAD-A65-ATG-F3: GCCCGCTTGAATTTAATGACGTTTGTTACTACG; GAD-A65-TGG-F4:GCCCGCTTGAATTTATGGACGTTTGTTACTACG; R4: CCGATGAACTAAATACAATATTGGGGGCGGTCCG.

[0027] T66 mutant library: GAD-T66-NDT-F1: CGCTTGAATTTAGCTNDTTTTGTTACTACGTGG; GAD-T66-VMA-F2: CGCTTGAATTTAGCTVMATTTGTTACTACGTGG; GAD-T66-ATG-F3: CGCTTGAATTTAGCTATGTTTGTTACTACGTGG; GAD-T66-TGG-F4: CGCTTGAATTTAGCTTGGTTTGTTACTACGTGG; R4: CCGATGAACTAAATACAATATTGGGGCGGTCCG.

[0028] F67 mutation library: GAD-F67-NDT-F1: TTGAATTTAGCTACGNDTGTTACTACGTGGATG; GAD-F67-VMA-F2: TTGAATTTAGCTACGVMAGTTACTACGTGGATG; GAD-F67-ATG-F3: TTGAATTTAGCTACGATGGTTACTACGTGGATG; GAD-F67-TGG-F4: TTGAATTTAGCTACGTGGGTTACTACGTGGATG; R4: CCGATGAACTAAATACAATATTGGGGCGGTCCG.

[0029] V68 mutation library: GAD-V68-NDT-F1: AATTTAGCTACGTTTNDTACTACGTGGATGGAG; GAD-V68-VMA-F2: AATTTAGCTACGTTTVMAACTACGTGGATGGAG; GAD-V68-ATG-F3: AATTTAGCTACGTTTATGACTACGTGGATGGAG; GAD-V68-TGG-F4: AATTTAGCTACGTTTTGGACTACGTGGATGGAG; R4: CCGATGAACTAAATACAATATTGGGGCGGTCCG.

[0030] G127 mutation library: GAD-G127-NDT-F1: GGCGTTTCTACTACANDTTCATCTGAAGCATGT; GAD-G127-VMA-F2: GGCGTTTCTACTACAVMATCATCTGAAGCATGT; GAD - G127 - ATG - F3: GGCGTTTCTACTACAATGTCATCTGAAGCATGT; GAD - G127 - TGG - F4: GGCGTTTCTACTACATGGTCATCTGAAGCATGT; R1: GGAGCGATAAAACCTCCAGAAGCAGCATCTACA.

[0031] S128 mutant library: GAD - S128 - NDT - F1: GTTTCTACTACAGGTNDTTCTGAAGCATGTATG; GAD - S128 - VMA - F2: GTTTCTACTACAGGTVMATCTGAAGCATGTATG; GAD - S128 - ATG - F3: GTTTCTACTACAGGTATGTCTGAAGCATGTATG; GAD - S128 - TGG - F4: GTTTCTACTACAGGTTGGTCTGAAGCATGTATG; R1: GGAGCGATAAAACCTCCAGAAGCAGCATCTACA.

[0032] S129 mutant library: GAD - S129 - NDT - F1: TCTACTACAGGTTCANDTGAAGCATGTATGCTT; GAD - S129 - VMA - F2: TCTACTACAGGTTCAVMAGAAGCATGTATGCTT; GAD - S129 - ATG - F3: TCTACTACAGGTTCAATGGAAGCATGTATGCTT; GAD - S129 - TGG - F4: TCTACTACAGGTTCATGGGAAGCATGTATGCTT; R1: GGAGCGATAAAACCTCCAGAAGCAGCATCTACA.

[0033] E130 mutant library: GAD - E130 - NDT - F1: ACTACAGGTTCATCTNDTGCATGTATGCTTGGT; GAD - E130 - VMA - F2: ACTACAGGTTCATCTVMAGCATGTATGCTTGGT; GAD-E130-ATG-F3: ACTACAGGTTCATCTATGGCATGTATGCTTGGT; GAD-E130-TGG-F4: ACTACAGGTTCATCTTGGGCATGTATGCTTGGT; R1: GGAGCGATAAAACCTCCAGAAGCAGCATCTACA.

[0034] C132 mutation library: GAD-C132-NDT-F1: GGTTCATCTGAAGCANDTATGCTTGGTGGACTA; GAD-C132-VMA-F2: GGTTCATCTGAAGCAVMAATGCTTGGTGGACTA; GAD-C132-ATG-F3: GGTTCATCTGAAGCAATGATGCTTGGTGGACTA; GAD-C132-TGG-F4: GGTTCATCTGAAGCATGGATGCTTGGTGGACTA; R1: GGAGCGATAAAACCTCCAGAAGCAGCATCTACA.

[0035] Q166 mutation library: GAD-Q166-NDT-F1: TTTAGTTCATCGGTTNDTGTGGTATGGGAGAAG; GAD-Q166-VMA-F2: TTTAGTTCATCGGTTVMAGTGGTATGGGAGAAG; GAD-Q166-ATG-F3: TTTAGTTCATCGGTTATGGTGGTATGGGAGAAG; GAD-Q166-TGG-F4: TTTAGTTCATCGGTTTGGGTGGTATGGGAGAAG; R1: GGAGCGATAAAACCTCCAGAAGCAGCATCTACA.

[0036] V168 mutation library: GAD-V168-NDT-F1: TCATCGGTTCAAGTGNDTTGGGAGAAGTTCGCA; GAD-V168-VMA-F2: TCATCGGTTCAAGTGVMATGGGAGAAGTTCGCA; GAD-V168-ATG-F3: TCATCGGTTCAAGTGATGTGGGAGAAGTTCGCA; GAD-V168-TGG-F4: TCATCGGTTCAAGTGTGGTGGGAGAAGTTCGCA; R1: GGAGCGATAAAACCTCCAGAAGCAGCATCTACA.

[0037] W169 mutant library: GAD-W169-NDT-F1: TCGGTTCAAGTGGTANDTGAGAAGTTCGCAAAC; GAD-W169-VMA-F2: TCGGTTCAAGTGGTAVMAGAGAAGTTCGCAAAC; GAD-W169-ATG-F3: TCGGTTCAAGTGGTAATGGAGAAGTTCGCAAAC; GAD-W169-TGG-F4: TCGGTTCAAGTGGTATGGGAGAAGTTCGCAAAC; R1: GGAGCGATAAAACCTCCAGAAGCAGCATCTACA.

[0038] I211 mutant library: GAD-I211-NDT-F1: ATTGGCGTCGTACCGNDTCTTGGAGTCACGTAT; GAD-I211-VMA-F2: ATTGGCGTCGTACCGVMACTTGGAGTCACGTAT; GAD-I211-ATG-F3: ATTGGCGTCGTACCGATGCTTGGAGTCACGTAT; GAD-I211-TGG-F4: ATTGGCGTCGTACCGTGGCTTGGAGTCACGTAT; R2: CCGTCTTTACCTAAACGCAAGAAATTGTAGTAC.

[0039] G213 mutant library: GAD-G213-NDT-F1: GTCGTACCGATTCTTNDTGTCACGTATACAGGG; GAD - G213 - VMA - F2: GTCGTACCGATTCTTVMAGTCACGTATACAGGG; GAD - G213 - ATG - F3: GTCGTACCGATTCTTATGGTCACGTATACAGGG; GAD - G213 - TGG - F4: GTCGTACCGATTCTTTGGGTCACGTATACAGGG; R2: CCGTCTTTACCTAAACGCAAGAAATTGTAGTAC.

[0040] T215 Mutation Library: GAD - T215 - NDT - F1: CCGATTCTTGGAGTCNDTTATACAGGGGGTTAC; GAD - T215 - VMA - F2: CCGATTCTTGGAGTCVMATATACAGGGGGTTAC; GAD - T215 - ATG - F3: CCGATTCTTGGAGTCATGTATACAGGGGGTTAC; GAD - T215 - TGG - F4: CCGATTCTTGGAGTCTGGTATACAGGGGGTTAC; R2: CCGTCTTTACCTAAACGCAAGAAATTGTAGTAC.

[0041] D246 Mutation Library: GAD - D246 - NDT - F1: ATTCCTATCCATGTANDTGCTGCTTCTGGAGGT; GAD - D246 - VMA - F2: ATTCCTATCCATGTAVMAGCTGCTTCTGGAGGT; GAD - D246 - ATG - F3: ATTCCTATCCATGTAATGGCTGCTTCTGGAGGT; GAD - D246 - TGG - F4: ATTCCTATCCATGTATGGGCTGCTTCTGGAGGT; R2: CCGTCTTTACCTAAACGCAAGAAATTGTAGTAC.

[0042] A248 Mutation Library: GAD-A248-NDT-F1: ATCCATGTAGATGCTNDTTCTGGAGGTTTTATC; GAD-A248-VMA-F2: ATCCATGTAGATGCTVMATCTGGAGGTTTTATC; GAD-A248-ATG-F3: ATCCATGTAGATGCTATGTCTGGAGGTTTTATC; GAD-A248-TGG-F4: ATCCATGTAGATGCTTGGTCTGGAGGTTTTATC; R2: CCGTCTTTACCTAAACGCAAGAAATTGTAGTAC.

[0043] S249 mutation library: GAD-S249-NDT-F1: CATGTAGATGCTGCTNDTGGAGGTTTTATCGCT; GAD-S249-VMA-F2: CATGTAGATGCTGCTVMAGGAGGTTTTATCGCT; GAD-S249-ATG-F3: CATGTAGATGCTGCTATGGGAGGTTTTATCGCT; GAD-S249-TGG-F4: CATGTAGATGCTGCTTGGGGAGGTTTTATCGCT; R2: CCGTCTTTACCTAAACGCAAGAAATTGTAGTAC.

[0044] S276 mutation library: GAD-S276-NDT-F1: AAGTCCATTAACGTGNDTGGACACAAGTATGGT; GAD-S276-VMA-F2: AAGTCCATTAACGTGVMAGGACACAAGTATGGT; GAD-S276-ATG-F3: AAGTCCATTAACGTGATGGGACACAAGTATGGT; GAD-S276-TGG-F4: AAGTCCATTAACGTGTGGGGACACAAGTATGGT; R3: CATCAGCAAGAATTTCGAATGCGTCCATTTCTC.

[0045] H278 mutation library: GAD-H278-NDT-F1: ATTAACGTGTCAGGANDTAAGTATGGTTTAGTT; GAD-H278-VMA-F2: ATTAACGTGTCAGGAVMAAAGTATGGTTTAGTT; GAD-H278-ATG-F3: ATTAACGTGTCAGGAATGAAGTATGGTTTAGTT; GAD-H278-TGG-F4: ATTAACGTGTCAGGATGGAAGTATGGTTTAGTT; R3: CATCAGCAAGAATTTCGAATGCGTCCATTTCTC.

[0046] K279 mutant library: GAD-K279-NDT-F1: AACGTGTCAGGACACNDTTATGGTTTAGTTTAC; GAD-K279-VMA-F2: AACGTGTCAGGACACVMATATGGTTTAGTTTAC; GAD-K279-ATG-F3: AACGTGTCAGGACACATGTATGGTTTAGTTTAC; GAD-K279-TGG-F4: AACGTGTCAGGACACTGGTATGGTTTAGTTTAC; R3: CATCAGCAAGAATTTCGAATGCGTCCATTTCTC.

[0047] R422 mutant library: GAD-R422-NDT-F1: GAAATCACAATCATGNDTATTGTTGTTAGAAAT; GAD-R422-VMA-F2: GAAATCACAATCATGVMAATTGTTGTTAGAAAT; GAD-R422-ATG-F3: GAAATCACAATCATGATGATTGTTGTTAGAAAT; GAD-R422-TGG-F4: GAAATCACAATCATGTGGATTGTTGTTAGAAAT; R5: GGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGC.

[0048] F465 mutant library: GAD-F465-NDT-F1:AAATACGACAATGGANDTCATCATTAAAAGCTT; GAD-F465-VMA-F2:AAATACGACAATGGAVMACATCATTAAAAGCTT; GAD-F465-ATG-F3:AAATACGACAATGGAATGCATCATTAAAAGCTT; GAD-F465-TGG-F4:AAATACGACAATGGATGGCATCATTAAAAGCTT; R5: GGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGC.

[0049] Table 1. First-round PCR reaction system

[0050] Table 2 Second round PCR reaction system

[0051] Table 3 PCR reaction procedure

[0052] After completing two rounds of PCR reactions, add to each reaction system Dpn Incubate 2 μL of enzyme I at 37°C for 3 hours, and then electroporate 1 μL of the resulting product into... E. coli BL21(DE3) competent cells were incubated upside down in a 37°C incubator for 12-16 hours. Once single clones grew out, the mutant library was constructed.

[0053] Example 3: High-throughput screening of a glutamate decarboxylase PmGAD gene mutant library and acquisition of engineered strains with single-point mutants. In this embodiment, the high-throughput screening method for the PmGAD gene mutant library of glutamate decarboxylase was ultraviolet spectrophotometry. L-glutamate is converted to GABA by glutamate decarboxylase, and the reaction equation is as follows: Figure 3 As shown, the pH gradually increases during the reaction, causing the colorimetric solution to change from yellow to purple, with a maximum absorption peak at 575 nm.

[0054] The specific methods and operating steps are as follows: (1) Preparation of bacterial cells The single colonies grown in Example 2 were picked up with a toothpick and cultured in a 96-well plate, which was designated as the mother plate. The plates were cultured at 37°C and 1200 rpm for 12 h with shaking. 120 μL of the overnight culture was transferred to a 96-well glycerol plate, and 80 μL of 50% glycerol was added and thoroughly mixed by pipetting. The plate was then capped and stored at -80°C. Simultaneously, 800 μL of TB+IPTG+Amp medium (0.1 M IPTG, 50 μg / mL Amp) was added to the 96-well plate, and the plates were cultured at 25°C and 1200 rpm for 12 h for protein expression.

[0055] (2) Whole-cell catalytic reaction Incubate 800 μL of bacterial culture at 4000 rpm for 5 min, discard the supernatant, and use 0.6% (v / v) chlorophenol red and bromocresol green sodium as indicators. Immediately add 100 μL of a solution containing the two indicators (1:1) and 10 g / L L-glutamic acid to each well, and react at 45℃ and 200 rpm for 30 min. Incubate at 4000 rpm for 4 min, collect the supernatant, and measure the OD using a microplate reader. 575 nm. Mutant strains with relatively high fluorescence intensity (i.e., relatively high enzyme activity) were screened for further screening.

[0056] (3) Obtaining single point mutant engineered strains OD 575 Strains with a value higher than that of the wild type under nm were sent for sequencing. Transformants that were correctly sequenced were identified as engineered strains of the glutamate decarboxylase PmGAD gene mutant.

[0057] Example 4: Preparation of engineered bacteria containing a two-site mutant of the glutamate decarboxylase PmGAD gene The double mutant in this embodiment is constructed by full plasmid PCR based on the corresponding single mutant, using the primers in Table 4. For example, based on the mutant F465L, the double mutant F465L / S276P is constructed by using the mutant primers S276P-F and S276P-R (Table 4). The PCR reaction system and procedure are shown in Tables 5 and 3.

[0058] Table 4 Primer sequences used for the two-site glutamate decarboxylase mutant

[0059] Table 5 PCR reaction system

[0060] Three double mutants were obtained using the above method and named mutant F465L / S276P, mutant F465L / S249F, and mutant S276P / S249F, respectively.

[0061] Example 5: Preparation of engineered bacteria with a three-point mutant of the glutamate decarboxylase PmGAD gene Based on the mutant F465L / S276P, a triple mutant was constructed by whole plasmid PCR using the mutant primers S249F-F and S249F-R (Table 4). For specific methods, please refer to Example 2. The primers used are shown in Table 4.

[0062] The above method yields three mutants, which are named mutants F465L / S276P / S249F.

[0063] Example 6: Obtaining crude glutamate decarboxylase solution and determining its activity (1) Obtaining crude enzyme solution Recombinant bacterial transformants containing the above-mentioned glutamate decarboxylase PmGAD gene or the recombinant plasmids obtained in Examples 3-5 were picked and added to 5 mL of LB liquid medium containing 50 μg / mL ampicillin sodium. The medium was incubated overnight at 37°C and 220 rpm for 12-16 hours with shaking. The transformants were then inoculated into LB liquid medium containing 50 μg / mL ampicillin sodium at a 1% (volume percentage) inoculation rate and cultured at 37°C for 5 hours. IPTG was added to a final concentration of 0.1 mmol / L, and expression was induced at 25°C and 220 rpm for 18 hours. The cells were then centrifuged at 4°C and 7000 rpm for 10 minutes to collect the bacterial cells. The collected bacterial cells were resuspended in PBS buffer (50 mM, pH 7.4) to obtain whole cells of glutamate decarboxylase PmGAD. The bacterial cells were then sonicated under ice bath conditions to obtain the sonicated sample. The sonicated sample was then centrifuged at 4°C and 7000 rpm for 10 minutes, and the supernatant (i.e., crude enzyme solution) was collected.

[0064] (2) Detection of glutamate decarboxylase PmGAD activity The enzymatic reaction system consisted of 1 mL of 100 g / L L-glutamate monohydrate, 1 mM PLP solution, and 100 μL crude enzyme solution; the reaction was carried out at pH 5.5, 37℃, and 220 rpm for 10 min.

[0065] To terminate the reaction: Dilute the reaction solution after the above reaction by 10 times with the mobile phase, boil for 2 minutes, centrifuge, and take 100 μL and mix it in 900 μL of mobile phase.

[0066] The sample was filtered through a 0.45 μm water membrane and detected by high-performance liquid chromatography (HPLC). Detection conditions: YMC-PackPro C18 column (4.6 × 250 mm, 5 μm), flow rate 1.2 mL / min; detection wavelength 210 nm; sample processing time 25 min; mobile phase: (potassium dihydrogen phosphate: sodium heptanesulfonate: methanol = 17:25:250, m / m / v). Under these conditions, the retention times for L-glutamic acid and γ-aminobutyric acid were 6.76 min and 8.12 min, respectively.

[0067] Enzyme activity is defined as the amount of enzyme required to convert 1 micromolar of substrate within 1 minute under specific conditions. One unit of activity (U) is defined as the amount of enzyme required to convert 1 micromolar of substrate within 1 minute.

[0068] Table 6 shows the enzyme activity detection results of single-site mutants with higher enzyme activity than wild-type glutamate decarboxylase, and Table 7 shows the enzyme activity detection results of multi-site glutamate decarboxylase mutants.

[0069] Table 6. Enzyme activity detection results of wild-type glutamate decarboxylase and single-site glutamate decarboxylase mutants.

[0070] Table 7 Results of enzyme activity assay for multi-site mutants

[0071] Example 7 Effect of different temperatures on the preparation of γ-aminobutyric acid In a 50 mL system, 100 g / L L-glutamate, 0.1 mM PLP, and 50 g / L wet bacterial cells expressing wild-type glutamate decarboxylase PmGAD were added sequentially, and the mixtures were incubated at 30-50 °C with stirring at 220 rpm. The conversion rates under different reaction conditions after 5 h of reaction are shown in Table 8 below. Table 8. Effect of different temperatures on L-glutamic acid conversion rate

[0072] Example 8 Effect of different pH values ​​on the preparation of γ-aminobutyric acid In a 50 mL system, 100 g / L L-glutamate, 0.1 mM PLP, and 50 g / L wet bacterial cells expressing wild-type glutamate decarboxylase PmGAD were added sequentially. The pH was adjusted to 3.0-6.0, and the system was incubated at 45 °C with stirring at 220 rpm to initiate the reaction. After 5 h of reaction, the conversion rates under different reaction conditions are shown in Table 9 below. Table 9. Effect of different pH values ​​on L-glutamic acid conversion rate

[0073] Example 9: Effect of different bacterial cell concentrations on the preparation of γ-aminobutyric acid In a 50 mL system, 100 g / L L-glutamate, 0.1 mM PLP, and 10-100 g / L wet bacterial cells expressing wild-type glutamate decarboxylase PmGAD were added sequentially. The pH was adjusted to 4.0, and the system was incubated at 45 °C with stirring at 220 rpm to initiate the reaction. After 5 h of reaction, the conversion rates under different reaction conditions are shown in Table 10 below. Table 10 Effect of different bacterial cell concentrations on L-glutamic acid conversion rate

[0074] Example 10 Effect of different substrate concentrations on the preparation of γ-aminobutyric acid In a 50 mL system, 100-600 g / L L-glutamate, 0.1 mM PLP, and 10 g / L wet bacterial cells expressing wild-type glutamate decarboxylase PmGAD were added sequentially. The pH was adjusted to 4.0, and the system was incubated at 45 °C with stirring at 220 rpm to initiate the reaction. After 5 h of reaction, the conversion rates under different reaction conditions are shown in Table 11 below. Table 11 Effect of different substrate concentrations on L-glutamate conversion

[0075] Example 11 Whole-cell catalytic synthesis of γ-aminobutyric acid (GABA) from glutamate decarboxylase PmGAD gene mutants (mutants F465L / S276P / S249F) (1L reaction system) In a 1L system, 600 g / L L-glutamic acid, 0.1 mM PLP, and 10 g / L of wet bacterial cells of mutant F465L / S276P were added sequentially. The pH was adjusted to 4.0, and the system was incubated at 45℃ with stirring at 220 rpm to initiate the reaction. After reacting for 5 h, 7 h, and 8 h, the conversion rate was measured to be 97.34% at 7 h and 99.49% at 8 h, producing 416.72 g / L γ-aminobutyric acid.

[0076] The results showed that the optimal mutant had better enzyme activity and whole-cell catalytic GABA production than the wild type (conversion rate of 74.38%). Furthermore, the whole-cell catalytic conversion rate of the 1L system using mutant F465L / S276P / S249F reached 99.21%, with a production efficiency of 52.09 g / L / h.

Claims

1. A glutamate decarboxylase PmGAD mutant, characterized in that, The mutant is obtained by mutating at least one of the following amino acids from SEQ ID NO:1: Phe at position 465, Ser at position 276, Ser at position 249, Glu at position 130, Thr at position 66, Gly at position 213, Ala at position 65, and Thr at position 215.

2. The glutamate decarboxylase PmGAD mutant according to claim 1, characterized in that, The mutant is based on SEQ ID NO:

1. Transform Phe at position 465 into Leu; or Mutate the 276th Ser bit to Pro; or Mutate the 249th Ser position to Phe; or Mutate Glu at position 130 to His; or Mutate the 66th Thr to Met; or Mutate the 66th Thr to Asp; or Mutate the 65th Ala to Ala (synonymous mutation); or Mutate Gly at position 213 to Arg; or Mutate Thr at position 215 to Phe; or Mutate the 215th Thr to Thr (synonymous mutation); or The Phe at position 465 was mutated to Ile.

3. The glutamate decarboxylase PmGAD mutant according to claim 1, characterized in that, The mutant is based on SEQ ID NO:

1. Mutate Phe at position 465 to Leu, and mutate Ser at position 276 to Pro; or Mutate Phe at position 465 to Leu, and mutate Ser at position 249 to Phe; or The 276th Ser was mutated to Pro, and the 249th Ser was mutated to Phe.

4. The glutamate decarboxylase PmGAD mutant according to claim 1, characterized in that, The mutant is based on SEQ ID NO:1, with Phe at position 465 mutated to Leu, Ser at position 276 mutated to Pro, and Ser at position 249 mutated to Phe.

5. The gene encoding the PmGAD mutant of glutamate decarboxylase according to any one of claims 1-4.

6. An expression vector carrying the gene of claim 5.

7. A recombinant strain comprising the expression vector as described in claim 6.

8. The use of the glutamate decarboxylase PmGAD mutant according to any one of claims 1-4 in the production of γ-aminobutyric acid.

9. A method for producing γ-aminobutyric acid, characterized in that, Includes the following steps: The glutamate decarboxylase PmGAD mutant of any one of claims 1-4 or the recombinant strain of claim 7 is added to a reaction system containing L-glutamate and pyridoxal 5-phosphate, and decarboxylation is carried out at pH 3.0-6.0 and 30-50℃ to generate γ-aminobutyric acid.

10. The method according to claim 9, characterized in that, The concentration of L-glutamic acid in the reaction system is 100-600 g / L, and the concentration of the recombinant strain is 10-100 g / L.

Images