CN116254253A - A kind of glutamic acid decarboxylase mutant obtained by DNA synthesis shuffling combination mutation and application - Google Patents

Abstract

The invention discloses a glutamic acid decarboxylase mutant enzyme whose thermostability is significantly improved through DNA synthesis shuffling and its application. The mutant enzyme is screened from a mutant library constructed by DNA synthesis shuffling, and the mutant library is composed of a random combination of mutation sites known to improve thermal stability and catalytic activity of glutamic acid decarboxylase (ie forward mutation). Through primary screening and secondary screening of this special library, a mutant enzyme D54A‑H181K‑D203E‑S325A with better thermal stability and catalytic activity than the wild-type enzyme was obtained: the half-inactivation temperature of the mutant enzyme was 71.23°C, which was higher than that of the wild-type enzyme. The wild-type enzyme (62.04°C) increased by 9.19°C; the catalytic efficiency increased by 59.5% compared with the wild-type enzyme. The mutant enzyme has better thermal stability and catalytic efficiency in the process of catalyzing the substrate to generate γ-aminobutyric acid, and is helpful for the large-scale biological preparation of γ-aminobutyric acid and its derivatives.

Description

A glutamic acid decarboxylase mutant obtained by DNA synthesis shuffling combinatorial mutation and applications

technical field

The invention relates to the technical field of molecular biology, in particular to a glutamic acid decarboxylase mutant obtained by DNA synthesis shuffling combination mutation and its application.

Background technique

Glutamate decarboxylase (glutamate decarboxylase, GAD; EC 4.1.1.15), under the action of pyridoxal-5-phosphate (PLP) coenzyme, can specifically catalyze the α-carboxyl decarboxylation reaction of L-Glu Generate γ-aminobutyric acid (γ-aminobutyric acid, referred to as GABA). GABA is a natural non-protein amino acid, which is widely distributed in animals, plants and microorganisms. As an important inhibitory neurotransmitter in the central nervous system of animals, GABA has been proved to be able to treat mental diseases (specific drugs for epilepsy and other diseases), anti-aging, regulate blood pressure, improve liver and kidney functions, and treat diabetes and other physiological functions. It has important functions in the fields of food and medicine. In 2009, GABA was also officially approved by the Ministry of Health of my country as "new resource food".

The preparation methods of GABA mainly include chemical synthesis, plant enrichment and microbial methods. Because the biosynthesis method has the advantages of mild reaction conditions, simple treatment procedures, high product yield and selectivity, energy saving and environmental protection, so the method of biological preparation of GABA has important industrial production application value. However, experiments have shown that the half-life of glutamic acid decarboxylase at 60°C is only 23.61min, which is very unfavorable for the application of GAD in the biological preparation of GABA, and its thermal stability needs to be further improved.

The patent application with publication number CN105462949A discloses a glutamic acid decarboxylase mutant and its preparation method and application. According to the comparison information with the GAD amino acid sequence of Thermococcus kodakarensis, the invention adopts the method of site-directed mutagenesis to introduce proline residues at the corresponding amino acid positions of Lactobacillus brevis GAD, and improves the thermal stability of GAD through rational design The half-inactivation temperature (T50 ¹⁵ ) of the mutant (G364P) is 61.6°C, which is 2.5°C higher than that of the wild-type enzyme; the half-life (t _1/2 ) at 55°C is 45.4min, which is longer than that of the wild-type enzyme 14.5min. Although the mutant obtained in the above experiments can increase the half-inactivation temperature and prolong the half-life compared with the wild-type glutamic acid decarboxylase, if it is to be better applied in actual production, the half-inactivation temperature and half-life of the above-mentioned mutant There is still room for further improvement.

The reaction rate increases with the increase of temperature, and for the industrial application and large-scale production of GABA, the enzyme needs to have high thermostability. DNA synthesis shuffling (synthetic shuffling) technology is a method for rapidly combining beneficial mutations, which can construct a mutation library combining different site substitutions. Through DNA synthesis shuffling, the positive mutations obtained in the previous screening were randomly combined in order to further improve the thermostability of glutamic acid decarboxylase.

Contents of the invention

The present invention uses synthetic shuffling technology to randomly combine the GAD forward mutations obtained in the previous research, thereby constructing a random combination mutation library, which is screened by a high-throughput color development method, and then its enzymatic properties are measured to finally obtain the improved thermal stability. mutant enzyme. This method can effectively increase the probability of successful mutation, and improve experimental efficiency and feasibility through high-throughput screening. The thermal stability of the obtained mutant enzyme is much higher than that of the wild-type enzyme, which improves the industrial application value of GAD and can convert the substrate into γ-aminobutyric acid more efficiently.

种突变，即进行1013次定点突变，而且每次定点突变后都需要进行突变酶的测序验证、重组表达和活性测定，必将耗费大量的时间和资源。而本发明只进行了3次PCR反应，再通过初筛和复筛就识别出了可以显著提高热稳定性和催化活性的突变位点组合方式。Before the disclosure of this mutation combination in the present invention, it could not be predicted from the existing data that this method can significantly improve the thermostability and catalytic activity of the enzyme at the same time - although a single mutation in it can help to improve the thermostability of the enzyme. However, due to the complex structure-activity relationship between protein structure and function, there is currently no reliable theory that can accurately analyze the synergistic effect of different amino acid mutations and the impact of different mutation combinations on enzyme stability and catalytic activity. If you try to discover the mutation by gradually superimposing point mutations and screening, theoretically you need to construct

One kind of mutation, that is, 1013 site-directed mutations, and after each site-directed mutation, sequencing verification, recombinant expression and activity measurement of the mutated enzyme are required, which will consume a lot of time and resources. However, in the present invention, only three PCR reactions are carried out, and the combination of mutation sites that can significantly improve thermal stability and catalytic activity is identified through primary screening and secondary screening.

The present invention firstly provides a glutamic acid decarboxylase mutant obtained by DNA synthesis shuffling combination mutation, which is obtained by introducing multiple sites from the wild-type glutamic acid decarboxylase from Lactobacillus brevis CGMCC NO.1306 derived from a mutation. The amino acid sequence of the wild-type glutamic acid decarboxylase is shown in SEQ ID NO.4, and the site mutation method of the glutamic acid decarboxylase mutant is: D54A-H181K-D203E-S325A.

D54A-H181K-D203E-S325A indicates that the amino acid at position 54 is mutated from aspartic acid to alanine, the amino acid at position 181 is mutated from histidine to lysine, and the amino acid at position 203 is mutated from aspartic acid It is glutamic acid, and the 325th amino acid is mutated from serine to alanine. The T50 ¹⁵ of the mutant enzyme is 71.23°C, which is 9.19°C higher than that of the wild-type enzyme (T50 ¹⁵ =62.04°C); k _cat /K _m value 59.5% higher than wild-type enzyme.

The wild-type glutamic acid decarboxylase is derived from Lactobacillus brevis CGMCC NO.1306, and the nucleotide sequence of the glutamic acid decarboxylase is shown in SEQ ID NO.3 (GenBank: GU987102.1).

The present invention further provides the application of the glutamic acid decarboxylase mutant in the biosynthesis of γ-aminobutyric acid.

The present invention further provides the gene encoding the glutamic acid decarboxylase mutant. Preferably, the nucleotide sequence of the gene is shown in SEQ ID NO.1.

The present invention also provides the application of the gene in the biosynthesis of γ-aminobutyric acid. The invention also provides a recombinant expression vector containing the gene. The present invention further provides a genetic engineering bacterium expressing the glutamic acid decarboxylase mutant. The present invention also provides the application of the genetically engineered bacteria in the biosynthesis of γ-aminobutyric acid.

Compared with the prior art, the present invention has the following beneficial effects:

The glutamic acid decarboxylase mutant of the present invention is composed of aspartic acid at position 54, histidine at position 181, and aspartic acid at position 203 from the wild-type glutamate decarboxylase of Lactobacillus brevis CGMCCNO.1306. Acid and 325-position serine were mutated into alanine, lysine, glutamic acid and alanine respectively. The T50 ¹⁵ of the mutant enzyme was 71.23°C, which was 9.19°C higher than that of the wild-type enzyme; k _cat /K _{The m} value increased by 59.5% compared with the wild-type enzyme. The mutant enzyme has better thermal stability and catalytic efficiency in the process of catalyzing the substrate to generate GABA, and is beneficial to the industrial production of GABA. The present invention utilizes synthetic shuffling technology to efficiently construct a mutation library theoretically including random combinations of all forward mutations through rapid random combination of existing forward mutations, and then obtains thermal stability and The catalytic activity of the mutant enzymes was superior to that of the wild-type enzyme.

Description of drawings

Figure 1 is a schematic diagram of the principle of the three-step method for synthesizing the target gene.

Fig. 2 is a schematic diagram of the principle of seamless cloning connection.

Figure 3 is the gel electrophoresis profile of the target gene synthesis and linearized vector; wherein, A is the gel electrophoresis profile of the first step of PCR synthesis of the target gene; B is the gel electrophoresis profile of the target gene and the linearized vector.

Fig. 4 is the SDS-PAGE diagram of the wild-type enzyme and the mutant enzyme.

Figure 5 shows the half-inactivation temperature (T50 ¹⁵ ) of the wild-type enzyme and the mutant enzyme at pH 4.8.

Fig. 6 is a half-life t _1/2 diagram of the wild-type enzyme and the mutant enzyme at different temperatures under the condition of pH 4.8; wherein, A is 60°C, and B is 70°C.

Detailed ways

In order to further illustrate the present invention, it is specifically described in conjunction with the following examples.

Example 1

1. Construction of mutant library

Forward mutants with improved enzymatic properties obtained by the mutation site selection research group in the previous research. Forward mutations were randomly introduced into the target gene by designing degenerate codons, as shown in Table 1.

Table 1 Mutations and degenerate codons used in the combinatorial library

The primer design required for the Synthetic shuffling technology is based on Ness et al. (Ness, J., Kim, S., Gottman, A. et al.Synthetic shuffling expands functional protein diversity by allowing amino acids to recombine independently. 2002).) The method described (the target gene is divided into small DNA fragments of equal length by primer design) is designed, as shown in Table 2.

Table 2 Primers required for Synthetic shuffling technology

Remarks: The underline indicates the mutation site, the one with F in the primer name indicates the upstream primer, and the one with R indicates the downstream primer.

The three-step method for synthesizing the target gene is shown in Figure 1.

(1) The first step of PCR uses the primers (F1-R18) in Table 2, and each set of PCR uses four adjacent oligonucleotide primers to assemble into a fragment with a length of about 200 bp. PCR amplification system: 25 μL PrimeSTAR Max Premix 2×, 0.5 μL inner primer (10 μM), 3 μL outer primer (10 μM) and 18 μL sterile water. PCR amplification conditions: denaturation at 94°C for 20s, annealing at 55°C for 15s, extension at 72°C for 30s, 30 cycles. The PCR products were verified by 1% agarose gel electrophoresis analysis, as shown in Figure 3A. 10 μL of the obtained 17 groups of different PCR products were mixed in 1.5 mL EP tubes and purified by a PCR product purification kit.

(2) The second step of assembly PCR: 17 groups of mixed gene fragments with an overlapping length of about 90 bp between two adjacent fragments in the first step of PCR were assembled into a complete target gene. PCR amplification system: 5 μL purified PCR product mixture, 20 μL PrimeSTAR Max Premix 2× and 25 μL sterile water. PCR amplification conditions: denaturation at 94°C for 30s, extension at 68°C for 120s, 20 cycles. The PCR product was purified by a purification kit.

(3) After the second step of assembly PCR is completed, the third step of PCR is performed according to the primers GAD-F and GAD-R to increase the number of complete target genes. PCR amplification system: 25 μL PrimeSTAR Max Premix 2×, 2 μL upstream primer (10 μM), 2 μL downstream primer (10 μM), 2 μL PCR product template (100 ng/μL) and 19 μL sterile water. PCR amplification conditions: denaturation at 98°C for 180s; denaturation at 98°C for 20s, annealing at 55°C for 15s, extension at 72°C for 120s, a total of 30 cycles; extension at 72°C for 420s. The PCR products were verified by 1% agarose gel electrophoresis analysis, as shown in Figure 3B.

(4) Linearization of pET-28a vector: Use primers pET-28a-F and pET-28a-R for vector PCR, and there is a 15-20bp homology arm between the linearized vector and the target gene. PCR amplification system: 25 μL PrimeSTAR Max Premix 2×; 2 μL upstream primer (10 μM), 2 μL downstream primer (10 μM), 1 μL plasmid template (100 ng/μL) and 20 μL sterile water. PCR amplification conditions: denaturation at 98°C for 180s; denaturation at 98°C for 20s, annealing at 55°C for 15s, extension at 72°C for 120s, a total of 30 cycles; extension at 72°C for 420s. After the PCR product was analyzed and verified by 1% agarose gel electrophoresis (as shown in FIG. 3B ), it was recovered by a gel recovery kit. The resulting gel-recovered product uses Dpn I enzyme to recognize and degrade the methylated parent template.

(5) Seamless cloning connection: the connection principle between the target gene and the linearized vector is shown in Figure 2. The amount of the linearized vector and the target gene added is 0.01-0.25 pmoles, and the optimal molar ratio of the linearized vector to the target gene is 1:2. Prepare a 10 μL recombination reaction system according to this condition: 4 μL (44 ng/μL) of pET-28a linearized vector, 1 μL (10 ⁶ ng/μL) of the target gene fragment, and 5 μL ClonExpress Mix 2×. Recombination conditions: ligate at 50°C for 30 minutes, and immediately place on ice to cool. The ligation product was transformed into Escherichia coli BL21(DE3) competent cells by heat shock method.

2. Induced expression of the mutant library.

The mutant library constructed using synthetic shuffling technology was picked from the corresponding master plate with a sterile toothpick and placed in a 48-well plate containing 50 μg/mL kanamycin in 4 mL LB liquid medium per well, cultured at 37 °C and 200 r/min 7h. Pipette 160 μL of bacterial solution from each well, mix with 40 μL of glycerol (60% by volume) and store in a -80°C refrigerator. Then, 200 μL of LB liquid medium (containing 0.5 mM IPTG) was added to the 48 deep-well plate, and cultured with shaking at 28° C. for 8 h to induce the expression of the target protein. After the induction, the orifice plate was centrifuged at 4000r/min for 15min to remove the supernatant and collect the bacteria. The bacteria were resuspended with 50mM PBS (pH 7.8), washed and transferred to a 96-well plate, and the supernatant was removed after centrifugation.

3. Screening of mutant library

The 96-deep-well plate was frozen at -80°C for 4 hours, then removed and incubated at 37°C for 30 minutes, and repeated freezing and thawing three times. Then, 200 μL of cell lysate (5 mg/mL lysozyme) was added to each well plate, and the cells were resuspended and placed in a constant temperature incubator at 37° C. and incubated at 100 r/min for 30 min. The 96-well plate was centrifuged at 4000r/min for 15min to collect the supernatant for color reaction.

The colorimetric reaction for mutant screening adopts the high-throughput screening method disclosed in the literature by Yu Kai et al.

4. Sequence the mutant enzyme gene obtained by screening

The high specific activity mutants obtained by screening in step (3) were cultured overnight at 37° C. and 200 r/min. Get 1ml bacterium liquid from it and send to Shanghai Sunny Biotechnology Co., Ltd. for sequencing, thereby clarifying the nucleotide sequence of this high specific activity mutant as shown in SEQ ID No.1, and the amino acid sequence of the enzyme encoded by it is as shown in SEQ ID As shown in No.2, it contains 4 amino acid mutation sites such as D54A, H181K, D203E and S325A, so it is named mutant enzyme D54A-H181K-D203E-S325A.

5. Expression and purification of wild-type and mutant enzymes

After activating the mutant enzyme D54A-H181K-D203E-S325A and the wild-type enzyme on the glycerin plate, pick a single colony and inoculate it into 5mL LB liquid medium containing 50μg/mL kanamycin, at 37°C, 200r/min Cultivate overnight under conditions, and then inoculate the overnight culture into 100 mL TB medium containing 50 μg/mL kanamycin (tryptone 12 g·L ^-1 , yeast extract 24g·L ^-1 , glycerol 4ml·L ^-1 , KH ₂ PO ₄ 17mmol·L ^-1 , K ₂ HPO ₄ 72mmol·L ^-1 ), cultivated at 37°C until the OD600 value was 0.6-0.8, and then added an appropriate amount of The volume of IPTG was adjusted to a final concentration of 0.5mmol/L, and then cultured at 28°C and 150r/min for 8 hours to collect the bacterial cells. Wash twice with phosphate buffer solution of pH 7.4, remove the medium and then resuspend the cells with 1/10 of the volume of the original fermentation broth, and ultrasonically disrupt the cells 90 times in an ice bath (300W, work for 3s, interval 6s), the crushed suspension was subpackaged and centrifuged at 13000r/min and 4°C for 25min to collect the supernatant, that is, the crude enzyme solution containing GAD. The obtained crude enzyme solution was separated and purified by Ni-NTA affinity chromatography. After loading, washing and eluting, the eluate is collected and dialyzed to remove small molecules to obtain pure enzyme. After appropriate dilution, the concentration of pure enzyme was determined by Coomassie brilliant blue method.

The buffer used was prepared as follows:

Lysis buffer (disruption buffer): 2mmol·L ^-1 potassium dihydrogen phosphate, 10mmol·L ^-1 disodium hydrogen phosphate, 2.7mmol·L ^-1 KCl, 137mmol·L ^-1 NaCl, pH 7.4, 10% (v /v) glycerol.

Wash buffer: 20mmol·L ^-1 Tris-Hcl, 300mmol·L ^-1 NaCl, 60mmol·L ^-1 imidazole, pH 7.8, 10% (v/v) glycerol.

Elution buffer: 20 mmol·L ⁻¹ Tris-Hcl, 300 mmol·L ⁻¹ NaCl, 400 mmol·L ⁻¹ imidazole, pH 7.8, 10% (v/v) glycerol.

The SDS-PAGE pictures of wild-type enzyme and mutant enzyme before and after purification are shown in Figure 4.

6. Determination of thermal stability of wild-type enzyme and mutant enzyme.

Determination of half-inactivation temperature T ₅₀ ¹⁵ : Take appropriate amount of pure enzyme and incubate at 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C for 15 minutes, then immediately transfer to ice Leave it on for 5min. The residual enzyme activities of enzymes after heat treatment at different temperatures were measured respectively.

Half-life (t _1/2 ) measurement conditions: take appropriate amount of pure enzyme and incubate at 60°C/70°C for different time, and measure the residual enzyme activity.

Method for measuring enzyme activity: Take 20 μL of pure enzyme and add 380 μL of substrate solution (0.2mol·L ^-1 acetic acid-sodium acetate buffer, 0.04mmol·L ^-1 PLP, 50mmol·L ^-1 substrate) preheated at 48°C L-MSG, pH 4.8), mixed quickly and reacted at 48°C for 10min, after the reaction was completed, put it into a metal bath at 100°C to inactivate for 10min to terminate the reaction, centrifuged, collected the supernatant, and used high performance liquid chromatography The amount of GABA produced by the reaction was measured to determine the specific activity of the enzyme. Pre-column derivatization of samples is required before HPLC determination. Derivatization method: 100 μL reaction solution, 100 μL 0.2 mol·L ^-1 sodium bicarbonate solution, 200 μL dansyl chloride-acetone solution (5 g/L), derivatize at 40°C for 2.5 hours in the dark. The derivatized samples were filtered through a 0.22 μm microporous membrane and injected.

The HPLC operating conditions are as follows: the chromatographic separation column is Hypersil ODS2 C18 (250mm×4.6mm) (Thermo Company), the ultraviolet detection wavelength is 254nm, the injection volume is 10 μ L, the column temperature is controlled at 25 ° C, the mobile phase A is methanol, and the mobile phase B Tetrahydrofuran:methanol:sodium acetate (0.05mol·L ^-1 , pH 6.2, 1:15:84, V/V/V). The gradient elution program is shown in Table 3.

Table 3 HPLC gradient elution program

T/min 0 5 20 twenty one 27 28 30 A% 20 20 50 100 100 20 20 B% 80 80 50 0 0 80 80

The experimental results are shown in Figure 5, the T ₅₀ ¹⁵ of the mutant enzyme D54A-H181K-D203E-S325A and the wild-type enzyme were 71.23°C and 62.04°C, respectively, and the mutant enzyme was 7.19°C higher than the wild-type enzyme; as shown in Figure 6, The half-life t _1/2 of the wild enzyme at 60°C is 23.61min, while the residual activity of the mutant enzyme is still 73.4% after heat treatment at 60°C for 125min, that is, its half-life at 60°C is greater than 125min; the half-life of the wild-type enzyme at 70°C is t _{1 /2} is 4.9min, while the t1 _/2 of the mutant enzyme is 32.4min, that is, the half-life of the wild-type enzyme at 70°C is 5.6 times higher than that of the wild-type enzyme.

This shows that the D54A-H181K-D203E-S325A four-site superimposed mutation enhances the thermostability of the enzyme, slows down the heat inactivation rate of the enzyme, and makes the mutant enzyme able to withstand higher temperatures and maintain activity. This feature will facilitate the application of the D54A-H181K-D203E-S325A mutant enzyme in the large-scale bioproduction of GABA.

7. Determination of Kinetic Parameters of Wild Enzyme and Mutant D54A-H181K-D203E-S325A

The initial reaction velocity at different substrate concentrations (L-MSG, 10-100 mmol·L ^-1 ) was measured. According to the Mie equation, plot 1/[S] against 1/[V] to calculate the corresponding K _m and V _max values. Then according to K _cat =V _max /[E ₀ ], [E ₀ ] is the initial concentration of the enzyme, and the unit is μmol/L, and the k _cat is calculated. The calculation results are shown in Table 4.

Table 4 Kinetic parameters of wild-type GAD and mutant enzyme D54A-H181K-D203E-S325A

K _m represents the affinity between the enzyme and the substrate. _kcat refers to the turnover number or catalytic constant, which indicates the number of molecules of substrate converted per molecule of enzyme or per enzyme active center per second when the enzyme is saturated with substrate. The larger the k _cat value, the higher the catalytic efficiency of the enzyme. k _cat /K _m is the apparent second-order rate constant of the product produced by the reaction between the enzyme and the substrate, which can be used to compare the catalytic efficiency of the enzyme.

It can be seen from the above table that the superposition mutation of the four sites D54A-H181K-D203E-S325A not only enhances the affinity of GAD to the substrate (K _m decreases from 27.45mM to 24.69mM), but also improves the catalytic activity of the enzyme active center (The k _cat value of the mutant enzyme was increased by 43% compared with that of the wild-type enzyme), and the overall effect increased the catalytic efficiency of the enzyme by 59.5%. This is also advantageous for the bioproduction of GABA using GAD.

Claims (8)

1. A glutamic acid decarboxylase mutant obtained by DNA synthesis shuffling combination mutation is characterized in that it is obtained by introducing multi-site mutations from wild-type glutamic acid decarboxylase from short lactobacillus (Lactobacillus brevis), The amino acid sequence of the wild-type glutamic acid decarboxylase is shown in SEQ ID NO.4, and the mutation site of the glutamic acid decarboxylase mutant is: D54A-H181K-D203E-S325A. 2. The application of the glutamic acid decarboxylase mutant as claimed in claim 1 in the biosynthesis of γ-aminobutyric acid. 3. A gene encoding the glutamic acid decarboxylase mutant according to claim 1. 4. The gene according to claim 3, wherein the nucleotide sequence is as shown in SEQ ID NO.1. 5. The application of the gene as claimed in claim 3 in the biosynthesis of γ-aminobutyric acid. 6. A recombinant expression vector containing the gene of claim 3. 7. A genetic engineering bacterium expressing the glutamic acid decarboxylase mutant described in claim 1. 8. The application of genetically engineered bacteria as claimed in claim 7 in the biosynthesis of γ-aminobutyric acid.

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