CN116083408A - High-activity L-glutamic acid alpha-decarboxylase mutant at neutral pH - Google Patents

Abstract

The invention discloses an L-glutamic acid alpha-decarboxylase mutant with high activity at neutral pH, belonging to the technical field of enzyme engineering. The invention generates GABA through one-step decarboxylation reaction, determines the site related to catalytic activity near a catalytic pocket through substrate butt joint, utilizes CAST to build a library NNK mutation, utilizes a constructed GABA sensor as a primary screening tool, utilizes fluorescence intensity as a screening basis, carries out combined mutation on the obtained sites of three libraries with effects (high fluorescence intensity), and then utilizes the sensor to carry out iterative screening twice, thus obtaining the L-glutamic acid alpha-decarboxylase mutant with improved enzyme activity under neutral pH environment. The specific enzyme activity of the mutant constructed by the invention is 64 times of that of the wild mutant under the neutral condition, which is beneficial to the industrialized production of GABA.

Description

High-activity L-glutamic acid alpha-decarboxylase mutant at neutral pH

Technical Field

The invention relates to an L-glutamic acid alpha-decarboxylase mutant with high activity at neutral pH, belonging to the technical field of enzyme engineering.

Background

Glutamate decarboxylase (glutamate decarboxylase, gad for short, EC 4.1.1.15) is a natural enzyme capable of catalyzing L-glutamic acid (L-Glu) to be converted into non-protein amino acid-gamma-aminobutyric acid (GABA) with high added value through decarboxylation reaction, and has been widely applied to industrial production of bulk chemicals gamma-aminobutyric acid. At present, the glutamic acid decarboxylase gradually replaces the traditional chemical method by the advantages of green environmental protection, mild reaction conditions, high safety coefficient and the like, so that the production of the gamma-aminobutyric acid accords with the sustainable development and green production concept. It has been found that most prokaryotic origin glutamate decarboxylases suffer from low catalytic activity at neutral pH. There have also been many attempts to modify it to improve its relevant properties. The locking effect of H465 on the active center is released by constructing C-terminal deletion (delta 452-456) mutants and terminal deletion (delta 465-466) mutants, and then the mutant E89Q obtained by means of directed evolution is subjected to point mutation, so that the enzymatic activity of GadB under neutral conditions is improved to a certain extent.

However, the problems of low catalytic efficiency at neutral pH and the like of the mutant still limit the further development and application of glutamate decarboxylase. Therefore, we have selected a new region by comparing GadB (delta 465-466) (M0) found by the previous people as the best starting mutant and further modifying E.coli source GadB by protein engineering, and then, we have screened out mutants with significantly improved enzyme activity by preliminary enzyme activity determination using a high throughput screening platform. This is of great importance for the production of GABA using GadB to catalyze glutamic acid.

Disclosure of Invention

Technical problems: aiming at the prior art difficulties and problems, the invention aims to provide a GadB mutant with improved glutamic acid catalyzing capability from E.coli.

The invention provides a glutamic acid decarboxylase mutant, which is characterized in that at least one of 51 st, 56 th, 68 th and 69 th amino acids is mutated on the basis of an amino acid sequence shown in SEQ ID NO. 1.

In one embodiment, the mutation is a mutation of tyrosine 51 to leucine, alanine 56 to proline, aspartic acid 68 to asparagine, and aspartic acid 69 to threonine, resulting in a mutant Y51L/A56P/D68N/D69T with the amino acid sequence shown in SEQ ID NO. 7.

The invention also provides genes encoding the mutants.

The invention also provides recombinant microorganisms expressing the mutants.

In one embodiment, the recombinant microorganism is recombinant E.coli.

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

The invention also provides a method for improving the catalytic capacity of glutamate decarboxylase in a neutral environment, wherein at least one amino acid of 51 st, 56 th, 68 th and 69 th is mutated.

In one embodiment, the method is to mutate tyrosine 51 to leucine and alanine 56 to proline and aspartic acid 68 to asparagine and aspartic acid 69 to threonine.

The invention also provides application of the mutant in gamma-aminobutyric acid production.

In one embodiment, the use is in the catalytic production of gamma-aminobutyric acid in a pH neutral environment using glutamic acid as a substrate.

In one embodiment, the pH neutral environment is an environment having a pH of 6 to 7.

The beneficial effects are that: the invention provides the amino acid sequence of the glutamic acid decarboxylase GadB, and the GadB mutant M3 is finally obtained by carrying out library building and screening on loop (50-69) near a catalytic pocket of an enzyme active center

(GadB-Y51L/A56P/D68N/D69T) the specific enzyme activity at 37 ℃ is 64 times that of M0. This is advantageous for industrial application in catalyzing the production of GABA from glutamic acid using this enzyme.

Drawings

Fig. 1: schematic diagram of the modified loop region around the GadB protein sequence and the GadB catalytic pocket.

Fig. 2: GABA sensor screening platform schematic.

Fig. 3: the first round of CAST library screening fluorescence intensity three rounds of transformation mutation screening fluorescence intensity summarization.

Fig. 4: the first round of CAST library screening enzyme activity comparison and three rounds of transformation mutant specific enzyme activity summarization.

Fig. 5: the course of the bioconversion reactions of M0 and M3 at initial pH6 and 7 was compared to the system pH change.

Detailed Description

Enzyme activity of glutamate decarboxylase (U): the unit enzyme activity is defined as the amount of enzyme required to catalyze the production of 1. Mu. Mol GABA per minute from glutamic acid at 37 ℃.

Specific enzyme activity of glutamate decarboxylase (U/mg): the enzyme activity of each milligram of glutamate decarboxylase.

The method for measuring the enzymatic activity of glutamate decarboxylase comprises the following steps: and calculating the specific enzyme activity corresponding to the glutamate decarboxylase in the system according to the amount of the pure enzyme reaction product measured by a High Performance Liquid Chromatograph (HPLC).

LB medium (per L): peptone 10g, yeast extract 5g, naCl 10g.

ZY5052 self-induction medium (per L): 10g of tryptone, 5g of yeast extract, 8.92g of disodium hydrogen phosphate dodecahydrate, 3.4g of potassium dihydrogen phosphate, 2.7g of ammonium chloride, 0.7g of sodium sulfate, 5g of glycerol, 0.5g of glucose and 2g of alpha-lactose.

Fluorescence value representation method: FI (fluorescence measurement)/OD ₆₀₀ 。

EXAMPLE 1 GadB Single Point mutant construction

After the glutamic acid decarboxylase (GadB) derived from escherichia coli (E.coli.JM 109) is docked with substrate glutamic acid, a Loop region which is possibly related to the catalytic capability of the enzyme near a catalytic pocket is selected for mutation library construction and screening. Ten libraries were designed using CAST method altogether as shown in table 1. Primers were designed for NNK mutation for each pool.

Cloning the gene shown in SEQ ID NO.2 (as shown in Table 2) with primers pet-GadB-D465-466-1, pet-GadB-D465-466-2, ligating the gene between araBAD promoter and λtL3 terminator of p15A plasmid (plasmids disclosed in Han L, liu X, cheng Z, cui W, guo J, yin J, zhou Z.construction and Application of aHigh-Throughput In Vivo Screening Platform for the Evolution of Nitrile Metabolism-Related Enzymes Based on a Desensitized Repressive Biosensor.Synth biol.2022Apr 15;11 (4): 1577-1587.) recombinant plasmids p15A-GadB were constructed using primers p15A-GadB-i1, p15A-GadB-i2, p15A-v1, p15A-v2 as shown in Table 2. Obtaining the p15A-GadB recombinant plasmid. Using the p15A-GadB plasmid as template and the primer sequence GadB-Nlopx-x (x is primer number), the amplification system is shown in Table 2, and the PCR amplification reaction conditions are 98 deg.C pre-denaturation for 3min,98 deg.C denaturation for 15s,55 deg.C annealing for 30s,72 deg.C extension for 1min and 72 deg.C extension for 5min, for 30 cycles. The PCR products were digested with DpnI digestive enzyme for 2-3h and purified to obtain individual pool single fragments.

Table 1 shows the design sites of the mutant library obtained

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

TABLE 2 primers

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TABLE 3 full plasmid PCR amplification reaction System

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TABLE 4 summary of mutant mutation sites obtained from CAST pool and combinatorial mutation

Example 2 screening of constructed CAST libraries Using GABA Sensors

Construction of competent cells containing GABA sensor: the sequence of the reporter gene, SEQ ID NO.8, containing the sensor transcription factor, promoter, was ligated between the T7 terminator and rop of plasmid pET24a (+) using the primers pET-GABA-i1, pET-GABA-i2 (plasmid disclosed in 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 2022Apr 15;11 (4): 1577-1587.), and transformed into E.coli JM109 to produce competent cells.

The fragment obtained by amplification in example 1 was transferred to competent cells containing GABA sensor, then substrate and inducer (0.1 mM arabinose, 20mM sodium glutamate, 50. Mu.g/mL kanamycin, 50. Mu.g/mL chloramphenicol) were added to LB medium, after culturing for 12-18 hours at 37℃colonies were selected under a blue light instrument, single colonies with stronger fluorescence were selected and inoculated to 96-well plate LB medium (500. Mu.L for each well, 50. Mu.g/mL for kanamycin final concentration, 34. Mu.g/mL for chloramphenicol final concentration) for 7-8 hours at 37℃and 300 rpm. The seed solution was transferred to 96-well LB medium (0.1 mM Ara, 20mM sodium glutamate, 50. Mu.g/mL kanamycin, 50. Mu.g/mL chloramphenicol) at 2% (v/v), and cultured at 37℃for 24 hours at 300 rpm. 200 mu L of bacterial liquid is sucked into an ELISA plate, and fluorescence detection is carried out by an ELISA instrument (detection condition: excitation wavelength 495nm and emission wavelength 525 nm).

The relative fluorescence value higher than that of the wild type was cultured in 10. Mu.L of the culture medium (final concentration of kanamycin: 50. Mu.g/mL; final concentration of chloramphenicol: 34. Mu.g/mL) at 37℃and 200rpm for 7-8 hours, and then transferred to 5mL of LB medium (0.1 mM Ara, 20mM sodium glutamate, 50. Mu.g/mL kanamycin; 50. Mu.g/mL chloramphenicol), and cultured at 200rpm for 24 hours at 37 ℃. 200 mu L of bacterial liquid is sucked into an ELISA plate, and fluorescence detection (detection conditions: excitation wavelength 495nm and emission wavelength 525 nm) is carried out by an ELISA instrument, and the detection result is shown in FIG. 3. And (3) preserving the mutant with higher relative fluorescence value obtained by detection, extracting plasmids for sequencing, purifying and measuring the enzyme activity of the pure enzyme, and comparing with M0. The obtained mutant site information is shown in table four. As shown in FIG. 3, FIG. 4, mutants 1-17,4-21, 10-6, FI/OD obtained by screening the CAST pool ₆₀₀ Significantly higher than M0, mutations of Y51T, A56P, D68N/D69T occurred, respectively, with the most effective mutant 10-6, the enzyme activity was increased by nearly 24-fold compared to M0.

Example 3 GadB combinatorial mutant construction and GABA sensor screening

According to the pure enzyme data corresponding to the high fluorescence mutants obtained by CAST library screening, the best three libraries (CAST 1, CAST4 and CAST 10) are selected for combined mutation screening. The first round of pure enzyme data obtained in example 2 was used to determine the screening order CAST10> CAST4> CAST1, the first 10-6 screening was designated M1, and M2 was obtained for CAST4 screening, and M3 was obtained for CAST1 screening. The optimal mutant plasmid obtained by each round of screening is used as a template, the primer sequences are shown in the following Table 2 GadB-Nlopx-x (x is the primer sequence number), the amplification system is shown in the following Table 3, the PCR amplification reaction conditions are 98 ℃ pre-denaturation for 3min,98 ℃ denaturation for 15s,55 ℃ annealing for 30s,72 ℃ extension for 1min and 72 ℃ extension for 5min, and 30 cycles are all performed. The PCR product was digested with DpnI digestive enzyme for 2-3 hours, and the combined mutant fragments were purified. The fragment was transferred to competent cells containing GABA sensor, then substrate and inducer (0.01 mM Ara, 20mM sodium glutamate, 50. Mu.g/mL kanamycin, 50. Mu.g/mL chloramphenicol) were added to LB medium, after culturing for 12-18 hours at 37℃colonies were selected under a blue light instrument, and single colonies with stronger fluorescence were selected and inoculated to 96-well plate LB medium (500. Mu.L for each well, 50. Mu.g/mL kanamycin final concentration, 34. Mu.g/mL chloramphenicol final concentration) for 7-8 hours at 300 rpm. The seed solution was transferred to 96-well LB medium (0.01 mM Ara, 20mM sodium glutamate, 50. Mu.g/mL kanamycin, 50. Mu.g/mL chloramphenicol) at 2% (v/v), and cultured at 37℃for 24 hours at 300 rpm. 200 mu L of bacterial liquid is sucked into an ELISA plate, and fluorescence detection is carried out by an ELISA instrument (detection condition: excitation wavelength 495nm and emission wavelength 525 nm).

The relative fluorescence value higher than that of the control was cultured in 10. Mu.L of the culture medium (kanamycin final concentration: 50. Mu.g/mL; chloramphenicol final concentration: 34. Mu.g/mL) at 37℃and 200rpm for 7-8 hours, and then transferred to 5mL of LB medium (0.01 mM Ara, 20mM sodium glutamate, 50. Mu.g/mL kanamycin, 50. Mu.g/mL; chloramphenicol) and cultured at 200rpm for 24 hours at 37 ℃. 200 mu L of bacterial liquid is sucked into an ELISA plate, and fluorescence detection (detection conditions: excitation wavelength 495nm and emission wavelength 525 nm) is carried out by an ELISA instrument, and the detection result is shown in FIG. 3. Mutants with high relative fluorescence values obtained by detection are stored and extracted to sequence plasmids, and the enzyme activity of the pure enzyme is purified and measured for comparison (see example 4).

The obtained mutant plasmids (1-17, 4-21, M0, M1, M2, M3) were used as templates, and PCR was performed using the primers P24a-mut-i1, P24a-mut-i2, P24a-mut-v1, and P24a-mut-v2 shown in Table 2, and the amplification systems were shown in Table 3. The amplification reaction conditions were 95℃pre-denaturation for 3min,95℃denaturation for 15s,55℃annealing for 30s,72℃extension for 1min20s,72℃extension for 5min for 30 cycles. The obtained fragments were size verified with nucleic acid gel and then sequenced by the company Jin Weizhi in su. Then amplifying the gene fragments of the GadB mutant from the plasmid which is sequenced normally and has mutation, wherein the primer sequences are shown in the table 2, the amplification system is shown in the table 3, and the PCR amplification reaction conditions are 98 ℃ pre-denaturation for 3min,98 ℃ denaturation for 15s,55 ℃ annealing for 30s,72 ℃ extension for 30s and 72 ℃ extension for 5min, and the total time is 30 cycles. Digesting the PCR product with DpnI digestive enzyme for 2-3h, purifying to obtain single fragment, assembling with general pET24a skeleton, incubating at 50deg.C for 30min with the assembly system of 4 μL, transferring to E.coli BL21, and pickingSingle colonies were cultured in 3mL of LB medium (final kanamycin concentration: 50. Mu.g/mL) at 37℃for 7-8 hours at 200 rpm. The seed solution was transferred to 100mL of ZY5052 medium (final kanamycin concentration: 50. Mu.g/mL) at 2% (v/v), and cultured at 37℃and 200rpm to OD ₆₀₀ To 0.6-0.8, isopropyl thiogalactoside (IPTG) was added at a final concentration of 0.05mM, the culture temperature was changed to 24℃and the expression was induced for 12-16h.

The mutant is purified by adopting an affinity chromatography method, and the purification column is a HisTrap HP 5mL column of GE company. The cells were collected by centrifugation at 10000rpm for 3min, resuspended in20 mL PBS buffer (pH 7.4) and sonicated in an ice-water mixture. The crushed solution was centrifuged at 12000rpm at 4℃for 30min, and the supernatant was filtered through a 0.22 μm organic filter. After equilibration with Binding buffer, the purification column is loaded, and then the foreign proteins are washed off with Binding buffer, and the target proteins are eluted with a gradient of elution buffer (Washing buffer) and collected. Protein concentration was quantified using Bradford protein concentration detection kit. The purification quality of the target protein is detected by SDS-PAGE, and the protein expressed by the wild type and the mutant thereof has single protein band and high purification quality after purification.

Pure enzyme reaction: a reaction system of 0.5mL, comprising 100mM sodium glutamate, 0.1mg/mL pure enzyme, pH7.4 PBS buffer, 0.25mM PLP, was reacted at 37℃for 20min, and the reaction was stopped at 100℃for 10 min. The sample was diluted 50-fold 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), and 1:1 were mixed) to obtain a sample, which was subjected to filtration through a 0.22 μm filter for 40 minutes, and then measured as a liquid phase. Determination of glutamate decarboxylase: GABA yield in the system was measured by HPLC, mobile phase A was 80% acetonitrile, mobile phase B was acetonitrile: 0.1M sodium acetate (3:97) (v: v), mobile phase A: B (5:95). The detection wavelength is 210nm, the flow rate is 0.6mL/min, the column temperature is 40 ℃, and the chromatographic column is a C18 column. Specific enzyme activity results of M0 and mutants are shown in FIG. four: the specific enzyme activities of the mutant M0 are 0.3U/mg, the specific enzyme activities of the mutants M1, 4-21 and 1-17 are 7.1U/mg, 4.2U/mg and 0.8U/mg respectively, and the specific enzyme activities of the mutants M2 and M3 are 12.7U/mg and 19.3U/mg respectively. The mutant site information obtained by combining the mutations is summarized in Table 4.

Namely, the specific enzyme activity of the mutant with high fluorescence intensity obtained by library building and screening is improved to different degrees, which shows that the selected locus has great effect on the catalytic activity of the glutamate decarboxylase.

Example 4 comparison of the course of bioconversion of M0 with M3 below initial pH7

Pure enzymes M0 and M3 were prepared in the same manner as in example 3, and the obtained pure enzyme solution was concentrated using a ultrafiltration tube having a molecular weight of 30kDa to obtain a pure enzyme solution having a concentration of 5mg/ml or more.

Pure enzymes were used for the conversion production of GABA. The 20mL reaction system comprises (by final concentration): 500mM sodium glutamate, 0.5mg/mL pure enzyme, diluted five times phosphate buffer (pH 6.0 or pH7.0, respectively), reacted at 37℃for 20min, 40min, 1h, 2h, 4h, 6h, 12h, respectively, terminated at 100℃for 10min and the GABA content was examined, and the conversion was calculated (conversion represents the ratio between the amount of the product substance produced and the amount of the initial substrate substance).

As shown in fig. 5, mutant M3 can maintain continuous and efficient catalytic activity under the condition of large system neutrality, and the conversion rate is 18 times higher than that of M0 in 12h and reaches 70% when the initial pH is 7; at an initial pH of 6, the conversion rate is improved by 4.4 times compared with M0 in 12 hours and reaches 83.4 percent.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (10)

1. The glutamic acid decarboxylase mutant is characterized in that at least one of 51 st, 56 th, 68 th and 69 th amino acids is mutated on the basis of the amino acid sequence shown in SEQ ID NO. 1.

2. The glutamate decarboxylase mutant according to claim 1, characterized by said mutation being the mutation of tyrosine 51 of the amino acid sequence shown in SEQ ID No.1 to leucine and alanine 56 to proline and aspartic acid 68 to asparagine and aspartic acid 69 to threonine.

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

4. A recombinant microorganism expressing the glutamate decarboxylase mutant of claim 1 or 2.

5. The recombinant microorganism according to claim 4, wherein the recombinant microorganism is a recombinant E.coli.

6. The recombinant microorganism according to claim 4, wherein the recombinant E.coli has pET-24a (+) as an expression vector and E.coli BL21 as an expression host.

7. A method for improving catalytic ability of glutamate decarboxylase under neutral environment is characterized in that at least one amino acid of 51 st, 56 th, 68 th and 69 th positions of the glutamate decarboxylase shown in SEQ ID NO.1 is mutated.

8. The method of claim 7, wherein the method comprises mutating tyrosine 51 to leucine and alanine 56 to proline and aspartic acid 68 to asparagine and aspartic acid 69 to threonine.

9. Use of the glutamate decarboxylase of claim 1 or 2 for the production of gamma-aminobutyric acid.

10. The use according to claim 9, wherein glutamic acid is used as a substrate for the catalytic production of gamma-aminobutyric acid in a pH neutral environment.

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