An L-glutamate α-decarboxylase mutant with high activity at neutral pH

Abstract

The present invention discloses an L-glutamate α-decarboxylase mutant with high activity at neutral pH, belonging to the technical field of enzyme engineering. Through a one-step decarboxylation reaction to generate GABA, the sites related to catalytic activity near the catalytic pocket were determined by substrate docking. Using CAST library NNK mutagenesis, with the constructed GABA sensor as a primary screening tool and fluorescence intensity as the screening basis, the sites of the three libraries with effective results (high fluorescence intensity) were subjected to combinatorial mutagenesis, and then the sensor was used for two iterative screenings to obtain an L-glutamate α-decarboxylase mutant with improved enzyme activity in a neutral pH environment. The mutant constructed in the present invention has a specific enzyme activity 64 times that of the wild type under neutral conditions, which is beneficial to the industrial production of GABA.

Description

Technical Field

[0001] The present invention relates to an L-glutamate α-decarboxylase mutant with high activity at neutral pH and belongs to the technical field of enzyme engineering. Background Art

[0002] Glutamate decarboxylase (abbreviated as Gad, EC 4.1.1.15) is a class of natural enzymes that can catalyze the conversion of L-glutamate (L-Glu) into a high-value non-protein amino acid - γ-aminobutyric acid (GABA) through a decarboxylation reaction and has been widely used in the industrial production of bulk chemical γ-aminobutyric acid. At present, glutamate decarboxylase gradually replaces the traditional chemical method with its advantages of environmental protection, mild reaction conditions, high safety factor, etc., making the production of γ-aminobutyric acid meet the concept of sustainable development and green production. It has been found that most glutamate decarboxylases derived from prokaryotes have the problem of low catalytic activity at neutral pH. Many studies have also tried to modify it to improve its related properties. For example, predecessors constructed C-terminal deletion (Δ452-456) mutants and terminal deletion (Δ465-466) mutants to relieve the locking effect of H465 on the active center. Subsequently, point mutations such as mutant E89Q obtained by directed evolution have all improved the enzyme activity of GadB under neutral conditions to a certain extent.

[0003] However, the problems such as low catalytic efficiency of the above mutants at neutral pH still limit the further development and application of glutamate decarboxylase. Therefore, through comparison, we further modified GadB derived from E. coli by protein engineering using the previously discovered GadB(Δ465-466)(M0) as the best starting mutant. We selected a brand-new region and preliminarily measured its enzyme activity using a high-throughput screening platform, and then screened out mutants with significantly improved enzyme activity. This is of great significance for using GadB to catalyze glutamate to produce GABA. Summary of the Invention

[0004] Technical Problem: Aiming at the existing technical difficulties and problems, the present invention aims to provide a GadB mutant derived from E. coli with improved ability to catalyze glutamate.

[0005] The present invention provides a glutamate decarboxylase mutant, which is a mutant of at least one amino acid at positions 51, 56, 68, and 69 based on the amino acid sequence shown in SEQ ID NO.1.

[0006] In one embodiment, the mutation is the substitution of tyrosine at position 51 with leucine, alanine at position 56 with proline, aspartic acid at position 68 with asparagine, and aspartic acid at position 69 with threonine, to obtain the mutant Y51L / A56P / D68N / D69T, whose amino acid sequence is shown in SEQ ID NO.7.

[0007] The present invention also provides a gene encoding the mutant.

[0008] The present invention also provides a recombinant microorganism expressing the mutant.

[0009] In one embodiment, the recombinant microorganism is recombinant Escherichia coli.

[0010] In one embodiment, the recombinant Escherichia coli uses pET-24a(+) as the expression vector and E. coli BL21 as the expression host.

[0011] The present invention also provides a method for improving the catalytic ability of glutamate decarboxylase in a neutral environment, which is to mutate at least one amino acid at positions 51, 56, 68, and 69.

[0012] In one embodiment, the method is to substitute tyrosine at position 51 with leucine, alanine at position 56 with proline, aspartic acid at position 68 with asparagine, and aspartic acid at position 69 with threonine.

[0013] The present invention also provides the application of the mutant in the production of γ-aminobutyric acid.

[0014] In one embodiment, the application uses glutamate as a substrate to catalytically produce γ-aminobutyric acid in a pH-neutral environment.

[0015] In one embodiment, the pH-neutral environment is an environment with a pH of 6-7.

[0016] Beneficial effects: The present invention provides the amino acid sequence of glutamate decarboxylase GadB, and by constructing a library and screening the loop (50-69) near the catalytic pocket of the enzyme active center, the GadB mutant M3

[0017] (GadB-Y51L / A56P / D68N / D69T) has a specific activity 64 times that of M0 at 37°C. This is beneficial for the industrial application of using this enzyme to catalyze glutamate to produce GABA. Brief Description of the Drawings

[0018] Figure 1 : Schematic diagram of the GadB protein sequence and the modified loop region near the GadB catalytic pocket.

[0019] Figure 2 : Schematic diagram of the GABA sensor screening platform.

[0020] Figure 3 : Summary of fluorescence intensities of the three-round modified mutant screening and the fluorescence intensity of the first-round CAST library screening.

[0021] Figure 4 : Summary of the specific enzyme activities of the three-round modified mutants and the comparison of the enzyme activities of the first-round CAST library screening.

[0022] Figure 5 : Comparison of the biotransformation reaction processes and the changes in the system pH between M0 and M3 at the initial pH values of 6 and 7. Specific implementation manners

[0023] Enzyme activity (U) of glutamate decarboxylase: The unit enzyme activity is defined as the amount of enzyme required to catalyze the formation of 1 μmol of GABA from glutamate per minute at 37°C.

[0024] Specific enzyme activity (U / mg) of glutamate decarboxylase: The enzyme activity possessed by each milligram of glutamate decarboxylase.

[0025] Method for measuring the enzyme activity of glutamate decarboxylase: Calculate the specific enzyme activity corresponding to glutamate decarboxylase in the system according to the amount of the product after the reaction of the pure enzyme measured by a high performance liquid chromatograph (HPLC).

[0026] LB medium (per liter): 10 g of peptone, 5 g of yeast extract, 10 g of NaCl.

[0027] ZY5052 autoinduction medium (per liter): 10 g of tryptone, 5 g of yeast extract, 8.92 g of disodium hydrogen phosphate dodecahydrate, 3.4 g of potassium dihydrogen phosphate, 2.7 g of ammonium chloride, 0.7 g of sodium sulfate, 5 g of glycerol, 0.5 g of glucose, 2 g of α-lactose.

[0028] Method for expressing fluorescence value: FI (measured fluorescence value) / OD 600 。

[0029] Example 1 Construction of GadB single-point mutants

[0030] After docking glutamate decarboxylase (GadB) from Escherichia coli (E.coli.JM109) with the substrate glutamate, select the Loop region near the catalytic pocket that may be related to the enzyme catalytic ability for mutant library screening. A total of ten libraries were designed using the CAST method as shown in Table 1. Design primers for NNK mutations for each library.

[0031] The gene shown in SEQ ID NO.2 (as shown in Table 2) was cloned using primers pet-GadB-D465-466-1 and pet-GadB-D465-466-2. The gene was ligated between the araBAD promoter and the λtL3 terminator of the p15A plasmid (the plasmid was disclosed in the paper Han L, Liu X, Cheng Z, Cui W, Guo J, Yin J, Zhou Z. Construction and Application of a High-Throughput In Vivo Screening Platform for the Evolution of Nitrile Metabolism-Related Enzymes Based on a Desensitized Repressive Biosensor. ACS Synth Biol. 2022 Apr 15;11(4):1577-1587.) to construct the recombinant plasmid p15A-GadB. The primers used were p15A-GadB-i1, p15A-GadB-i2, p15A-v1, and p15A-v2, as shown in Table 2. The p15A-GadB recombinant plasmid was obtained. Using the p15A-GadB plasmid as a template, the primer sequences GadB-Nloopx-x (x is the primer number) were used, as shown in Table 2. The amplification system is shown in Table 3. The PCR amplification reaction conditions were pre-denaturation at 98°C for 3 min, denaturation at 98°C for 15 s, annealing at 55°C for 30 s, extension at 72°C for 1 min 45 s, and extension at 72°C for 5 min, for a total of 30 cycles. The PCR product was digested with DpnI restriction enzyme for 2 - 3 h, and each single fragment of the library was purified.

[0032] Table 1 Design Sites of the Obtained Mutation Libraries

[0033] CAST round Mutation site 1 L50, Y51 2 L52, D53 3 G54, 55N 4 A56, R57 5 Q58, N59 6 L60, A61 7 T62, F63 8 C64, Q65 9 T66, W67 10 D68N, D69T

[0034] Table 2 Primers

[0035]

[0036]

[0037] Table 3 Whole Plasmid PCR Amplification Reaction System

[0038]

[0039]

[0040] Table 4 Summary of Mutation Sites of the CAST Library and Mutants Obtained by Combinatorial Mutations

[0041]

[0042] Example 2 Screening the constructed CAST library using a GABA sensor

[0043] Constructing competent cells containing a GABA sensor: Using primers pet-GABA-i1 and pet-GABA-i2, clone the sequence SEQ ID NO.8 containing the sensor transcription factor, promoter, and reporter gene and ligate it between the T7 terminator and rop of plasmid pET24a(+) (the plasmid is disclosed in the paper Han L, Liu X, Cheng Z, Cui W, Guo J, Yin J, Zhou Z. Construction and Application of a High-Throughput In Vivo Screening Platform for the Evolution of Nitrile Metabolism-Related Enzymes Based on a Desensitized Repressive Biosensor. ACS Synth Biol. 2022 Apr 15;11(4):1577-1587.). Transform this plasmid into Escherichia coli JM109 to prepare competent cells.

[0044] Transfer the fragment amplified in Example 1 into the competent cells containing the GABA sensor. Then, add the substrate and inducer (0.1 mM arabinose, 20 mM sodium glutamate, 50 μg / mL kanamycin, 50 μg / mL chloramphenicol) to the LB medium. After culturing at 37°C for 12 - 18 h, select the colonies under a blue light instrument, and pick the single colonies with stronger fluorescence and transfer them to the LB medium in a 96-well plate (add 500 μL to each well, final concentration of kanamycin is 50 μg / mL, final concentration of chloramphenicol is 34 μg / mL) and culture at 37°C and 300 rpm for 7 - 8 h. Transfer the seed liquid at 2% (v / v) to the LB medium in a 96-well plate (0.1 mM Ara, 20 mM sodium glutamate, 50 μg / mL kanamycin, 50 μg / mL chloramphenicol) and culture at 37°C and 300 rpm for 24 h. Pipette 200 μL of the bacterial liquid into an enzyme-linked immunosorbent assay (ELISA) plate and perform fluorescence detection using an enzyme-labeled instrument (detection conditions: excitation wavelength 495 nm, emission wavelength 525 nm).

[0045] Transfer 10 μL of the bacterial solution with a relative fluorescence value higher than that of the wild type into 3 mL of LB medium (final concentration of kanamycin: 50 μg / mL, final concentration of chloramphenicol: 34 μg / mL), and culture at 37 °C and 200 rpm for 7 - 8 h. Then transfer it to 5 mL of LB medium (0.1 mM Ara, 20 mM sodium glutamate, 50 μg / mL kanamycin, 50 μg / mL chloramphenicol), and culture at 37 °C and 200 rpm for 24 h. Pipette 200 μL of the bacterial solution into an ELISA plate, and perform fluorescence detection with an ELISA reader (detection conditions: excitation wavelength 495 nm, emission wavelength 525 nm). The detection results are as Figure 3 shown. Preserve the mutants with relatively high fluorescence values detected, extract plasmids for sequencing, purify and measure the enzyme activity of the pure enzyme, and compare it with M0. The mutant site information obtained is shown in Table 4. As Figure 3 , Figure 4 shown, the mutants 1 - 17, 4 - 21, 10 - 6, FI / OD 600 obtained by screening the CAST library are significantly higher than M0, and have mutations of Y51T, A56P, D68N / D69T respectively. Among them, the most obvious mutant 10 - 6 has an enzyme activity increased by nearly 24 times compared with M0.

[0046] Example 3 Construction of GadB combinatorial mutants and screening of GABA sensors

[0047] Based on the pure enzyme data corresponding to the highly fluorescent mutants screened from the CAST library, select the best three libraries (CAST1, CAST4, and CAST10) for combinatorial mutagenesis screening. Determine the screening order as CAST10 > CAST4 > CAST1 according to the pure enzyme data obtained in Example 2. Name the mutants obtained from the first screening at 10-6 as M1, and then screen M2 from CAST4 and M3 from CAST1 in the same way. Use the plasmid of the best mutant obtained in each round of screening as a template, and the primer sequences are shown in Table 2 GadB-Nloopx-x (x is the primer number). The amplification system is shown in Table 3. The PCR amplification reaction conditions are pre-denaturation at 98°C for 3 min, denaturation at 98°C for 15 s, annealing at 55°C for 30 s, extension at 72°C for 1 min 45 s, and final extension at 72°C for 5 min, for a total of 30 cycles. Digest the PCR product with DpnI for 2 - 3 h and purify the combinatorial mutant fragment. Transfer the fragment into competent cells containing the GABA sensor, and then add the substrate and inducer (0.01 mM Ara, 20 mM sodium glutamate, 50 μg / mL kanamycin, 50 μg / mL chloramphenicol) to the LB medium. After culturing at 37°C for 12 - 18 h, select colonies under a blue light instrument, and select single colonies with stronger fluorescence and transfer them to the 96-well plate LB medium (add 500 μL to each well, final concentration of kanamycin 50 μg / mL, final concentration of chloramphenicol 34 μg / mL) and culture at 37°C and 300 rpm for 7 - 8 h. Transfer the seed liquid to the 96-well plate LB medium (0.01 mM Ara, 20 mM sodium glutamate, 50 μg / mL kanamycin, 50 μg / mL chloramphenicol) at 2% (v / v) and culture at 37°C and 300 rpm for 24 h. Pipette 200 μL of the bacterial liquid into the microplate and perform fluorescence detection with a microplate reader (detection conditions: excitation wavelength 495 nm, emission wavelength 525 nm).

[0048] Transfer 10 μL of the bacterial liquid with a relative fluorescence value higher than the control to 3 mL of LB medium (final concentration of kanamycin 50 μg / mL, final concentration of chloramphenicol 34 μg / mL) and culture at 37°C and 200 rpm for 7 - 8 h, then transfer it to 5 mL of LB medium (0.01 mM Ara, 20 mM sodium glutamate, 50 μg / mL kanamycin, 50 μg / mL chloramphenicol) and culture at 37°C and 200 rpm for 24 h. Pipette 200 μL of the bacterial liquid into the microplate and perform fluorescence detection with a microplate reader (detection conditions: excitation wavelength 495 nm, emission wavelength 525 nm). The detection results are as Figure 3 shown. Preserve the mutants with high relative fluorescence values obtained from the detection, extract the plasmids for sequencing, and purify and measure the pure enzyme activity for comparison with the control (see Example 4).

[0049] Using the obtained mutant plasmids (1-17, 4-21, M0, M1, M2, M3) as templates respectively, PCR was performed with the primers P24a-mut-i1, P24a-mut-i2, P24a-mut-v1, P24a-mut-v2 shown in Table 2, and the amplification system was as shown in Table 3. The amplification reaction conditions were pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 55°C for 30 s, extension at 72°C for 1 min 20 s, extension at 72°C for 5 min, for a total of 30 cycles. The obtained fragments were verified for size by nucleic acid gel, and then sequenced by Suzhou Genewiz Co., Ltd. Then, the plasmid with normal sequencing and mutations was used to amplify the gene fragment of the GadB mutant. The primer sequences were as shown in Table 2, the amplification system was as shown in Table 3, and the PCR amplification reaction conditions were pre-denaturation at 98°C for 3 min, denaturation at 98°C for 15 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, extension at 72°C for 5 min, for a total of 30 cycles. The PCR product was digested with DpnI digestion enzyme for 2-3 h, purified to obtain a single fragment, and assembled with the universal pET24a backbone. The assembly system was 4 μL, incubated at 50°C for 30 min, and then transformed into E. coli BL21. Single colonies were picked into 3 mL LB medium (kanamycin final concentration 50 μg / mL) and cultured at 37°C and 200 rpm for 7-8 h. The seed solution was transferred to 100 mL ZY5052 medium (kanamycin final concentration 50 μg / mL) at 2% (v / v) and cultured at 37°C and 200 rpm until OD 600 reached 0.6-0.8, isopropyl β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.05 mM was added, and the culture temperature was changed to 24°C for induction expression for 12-16 h.

[0050] The above mutant was purified by affinity chromatography. The purification column was a HisTrap HP 5 mL column from GE. The bacterial cells were collected by centrifugation at 10000 rpm for 3 min, resuspended in 20 mL PBS buffer (pH 7.4), and ultrasonically disrupted in an ice-water mixture. The disrupted solution was centrifuged at 4°C and 12000 rpm for 30 min, and the supernatant was filtered through a 0.22 μm organic filter membrane. After the purification column was equilibrated with the binding buffer, the sample was loaded, and then the impurity proteins were washed away with the binding buffer. The target protein was eluted with a gradient of the elution buffer (Washing buffer) and collected. The protein concentration was quantified using a Bradford protein concentration detection kit. SDS-PAGE was used to detect the purification quality of the target protein. It can be seen that the proteins expressed by the wild type and its mutants have a single protein band after purification and high purification quality.

[0051] Pure enzyme reaction: The reaction system is 0.5 mL, including 100 mM sodium glutamate, 0.1 mg / mL pure enzyme, pH 7.4 PBS buffer, 0.25 mM PLP. React at 37 °C for 20 min and terminate the reaction at 100 °C for 10 min. Dilute the sample 50 times and derivatize it with an equal volume of derivatizing agent (Derivatizing agent A: triethylamine acetonitrile solution (14:86) (v:v); Derivatizing agent B: PITC acetonitrile solution (1:84) (v:v), mix the two in a ratio of 1:1) for 40 min. After passing through a 0.22-μm filter membrane, it is used as the sample for liquid phase determination. Determination of glutamate decarboxylase: Detect the GABA production in the system by HPLC. Mobile phase A is 80% acetonitrile, and mobile phase B is acetonitrile: 0.1 M sodium acetate (3:97) (v:v). Mobile phase A:B is (5:95). The detection wavelength is 210 nm, the flow rate is 0.6 mL / min, the column temperature is 40 °C, and the chromatographic column is a C18 column. The specific enzyme activity results of M0 and mutants are as Figure 4 shown: The specific enzyme activity of mutant M0 is 0.3 U / mg, and the specific enzyme activities of mutants M1, 4-21, and 1-17 are 7.1 U / mg, 4.2 U / mg, and 0.8 U / mg respectively. The specific enzyme activities of M2 and M3 are 12.7 U / mg and 19.3 U / mg respectively. The summary of the mutant site information obtained by combined mutation is shown in Table 4.

[0052] That is, the specific enzyme activities of the mutants with high fluorescence intensity obtained by library construction and screening have been improved to varying degrees, indicating that the sites we selected play a significant role in the catalytic activity of glutamate decarboxylase.

[0053] Example 4 Comparison of the Biotransformation Processes of M0 and M3 at an Initial pH below 7

[0054] Prepare the pure enzymes of M0 and M3 according to the method of Example 3, and concentrate the obtained pure enzyme solution using an ultrafiltration tube with a molecular weight cut-off of 30 KDa to obtain a pure enzyme solution with a concentration of more than 5 mg / ml.

[0055] Use the pure enzyme for the production of GABA by transformation. The 20 mL reaction system includes (at the final concentration): 500 mM sodium glutamate, 0.5 mg / mL pure enzyme, phosphate buffer diluted five times (pH 6.0 or pH 7.0 respectively). React at 37 °C on a shaker for 20 min, 40 min, 1 h, 2 h, 4 h, 6 h, and 12 h respectively. Terminate the reaction at 100 °C for 10 min and detect the GABA content and calculate the conversion rate (the conversion rate represents the ratio of the amount of product formed to the amount of initial substrate).

[0056] As Figure 5As shown, mutant M3 can maintain continuous and efficient catalytic activity under neutral conditions in a large system. When the initial pH is 7, the conversion rate within 12 hours is 18 times higher than that of M0, reaching 70%; when the initial pH is 6, the conversion rate within 12 hours is 4.4 times higher than that of M0, reaching 83.4%.

[0057] Although the present invention has been disclosed above in preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Glutamate decarboxylase mutant, characterized in that, Based on the amino acid sequence shown in SEQ ID NO.1, the alanine at position 56 was mutated to proline.

2. Glutamic acid decarboxylase mutant, characterized in that, Based on the amino acid sequence shown in SEQ ID NO.1, the aspartic acid at position 68 was mutated to asparagine, and the aspartic acid at position 69 was mutated to threonine.

3. Glutamate decarboxylase mutant, characterized in that, Based on the amino acid sequence shown in SEQ ID NO.1, the alanine at position 56 was mutated to proline, the aspartic acid at position 68 was mutated to asparagine, and the aspartic acid at position 69 was mutated to threonine.

4. Glutamate decarboxylase mutant, characterized in that, Based on the amino acid sequence shown in SEQ ID NO.1, the tyrosine at position 51 was mutated to leucine, the alanine at position 56 was mutated to proline, the aspartic acid at position 68 was mutated to asparagine, and the aspartic acid at position 69 was mutated to threonine.

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

6. A recombinant microorganism expressing the glutamate decarboxylase mutant according to any one of claims 1 to 4.

7. The recombinant microorganism according to claim 6, wherein, The recombinant microorganism is recombinant Escherichia coli.

8. The recombinant microorganism according to claim 7, characterized in that, The recombinant Escherichia coli uses pET-24a(+) as the expression vector and E. coli BL21 as the expression host.

9. A method for improving the catalytic ability of glutamate decarboxylase in a neutral environment, characterized in that, Comprising: (a) Mutating the aspartic acid at position 68 of the glutamate decarboxylase shown in SEQ ID NO.1 to asparagine, and mutating the aspartic acid at position 69 to threonine; (b) Mutating the alanine at position 56 of the glutamate decarboxylase shown in SEQ ID NO.1 to proline, mutating the aspartic acid at position 68 to asparagine, and mutating the aspartic acid at position 69 to threonine; (c) Mutating the tyrosine at position 51 of the glutamate decarboxylase shown in SEQ ID NO.1 to leucine, mutating the alanine at position 56 to proline, mutating the aspartic acid at position 68 to asparagine, and mutating the aspartic acid at position 69 to threonine.

10. Use of the glutamate decarboxylase mutant according to any one of claims 1 to 4 in the production of γ-aminobutyric acid.

11. The application according to claim 10, characterized in that, Using glutamate as a substrate to catalytically produce γ-aminobutyric acid in a neutral pH environment.

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