Process for the enzymatic preparation of r-3-aminobutyric acid

Abstract

This invention provides an enzymatic method for the preparation of R-3-aminobutyric acid (GABA), belonging to the field of biochemistry. Using crotonic acid as a substrate and a recombinant aspartate enzyme mutant as a biocatalyst, an enzymatic reaction is carried out under suitable temperature and pH control to efficiently and stereoselectively prepare the product R-3-aminobutyric acid. This method utilizes *E. coli* as a host for heterologous expression of recombinant aspartate enzyme. The whole cells of the expression host are used as crude enzyme for the enzymatic reaction. After extraction and purification, high-purity R-3-aminobutyric acid is obtained, with a conversion rate exceeding 99% and a product purity >99% and >99.9% ee. This method features high conversion and high yield, and is easily industrialized.

Description

Technical Field

[0001] This invention relates to an enzymatic preparation process for R-3-aminobutyric acid, belonging to the field of biochemical technology. Technical Background

[0002] Dolutegravir (DTG) is a novel anti-HIV drug approved by the U.S. Food and Drug Administration in 2013 for use in combination with other antiretroviral drugs to treat HIV-1 infection in humans. R-3-aminobutyric acid (CAS: 3775-73-3) is a stereoselective β-amino acid that serves as a precursor to the pharmaceutical intermediate R-3-aminobutanol, which is then reduced in one step to R-3-aminobutanol, a key intermediate in the production of dolutegravir.

[0003] Currently, the main methods for preparing R-3-aminobutyric acid (GABA) include chemical synthesis and biological methods. Chemical synthesis is complex and costly, has limited stereoselectivity, involves high-temperature and high-pressure operations, and causes significant environmental pollution, making it unsuitable for large-scale industrial production. Therefore, enzymatic methods for preparing GABA are gradually becoming the focus of industrialization.

[0004] Enzymatic methods have advantages such as high substrate specificity, mild reaction conditions, and strong stereoselectivity, but for a long time, the selection of enzyme sequences has limited the substrate conversion rate. Aspartate aminotransferase (Asp, EC4.3.1.1) is one of the three aminotransferase families. It catalyzes the reversible reaction of L-aspartic acid to produce ammonia and fumaric acid, and plays an important role in microbial nitrogen metabolism. It is also an industrial precursor for the synthesis of food additives and artificial sweeteners. The sources of aspartate aminotransferase currently studied mainly include Escherichia coli, Pseudomonas fluorescens, and Bacillus. Vogel et al. [ChemCatChem, 2014, 6(4), 965–968] modified the substrate binding pocket of AspB from Bacillus sp. YM55-1 to adapt to crotonic acid by enzyme engineering. The mutant enzyme BSASP-C6 catalyzes the conversion of crotonic acid and ammonia to R-3-aminobutyric acid (GABA) at a substrate concentration of 300 mM, achieving a final conversion rate of approximately 60% after 100 hours. This process is time-consuming and yields a low conversion rate, making it unsuitable for industrial production.

[0005] Therefore, finding more efficient enzyme sequences to improve substrate conversion, and developing efficient and low-cost process routes are key steps in the enzymatic preparation of R-3-aminobutyric acid and its industrialization. Summary of the Invention

[0006] To overcome the aforementioned technical deficiencies, the purpose of this invention is to provide an amino acid sequence of aspartic acid enzyme and a process for preparing R-3-aminobutyric acid (R-3-aminobutyric acid) using this enzyme, thereby further promoting the industrial production of R-3-aminobutyric acid. Using crotonic acid as a substrate and a recombinant aspartic acid enzyme mutant as a biocatalyst, an enzymatic reaction is carried out under suitable temperature and pH control to efficiently and stereoselectively prepare the product R-3-aminobutyric acid. This method utilizes *E. coli* as a host for heterologous expression of the recombinant aspartic acid enzyme. The whole cells of the expression host are used as the crude enzyme for the enzymatic reaction. After extraction and purification, a high-purity R-3-aminobutyric acid product is obtained, with a conversion rate exceeding 99% and a product purity >99% and >99.9% ee. This invention's method features high conversion and high yield, and is easily industrialized.

[0007] The present invention discloses an aspartic acid enzyme mutant, which is obtained by single or double mutation of leucine at position 165 or methionine at position 387 of the amino acid sequence of wild-type aspartic acid enzyme derived from Pseudomonas flavescens (shown in SEQ ID NO.2).

[0008] Furthermore, the mutant is obtained by mutating leucine at position 165 of the amino acid sequence shown in SEQ ID NO.2 to proline, and the mutant aspartate enzyme sequence is shown in SEQ ID NO.3.

[0009] Furthermore, the mutant is obtained by mutating methionine at position 387 of the amino acid sequence shown in SEQ ID NO.2 to alanine, and the mutant aspartate enzyme sequence is shown in SEQ ID NO.4.

[0010] Furthermore, the mutant is obtained by mutating leucine at position 165 of the amino acid sequence shown in SEQ ID NO.2 to proline, and simultaneously mutating methionine at position 387 to alanine, thus obtaining a two-point combination mutant; that is, mutating methionine at position 387 of the amino acid sequence shown in SEQ ID NO.3 to alanine, and the mutant aspartate enzyme sequence is shown in SEQ ID NO.5.

[0011] The present invention also provides a recombinant expression vector constructed from the aforementioned aspartic acid enzyme mutant sequence.

[0012] The present invention also provides a recombinant genetically engineered bacterium prepared by transformation of the aforementioned recombinant expression vector.

[0013] Furthermore, the present invention also provides an application of the preparation of R-3-aminobutyric acid from crotonic acid using the aspartic acid mutant.

[0014] Furthermore, this application uses crotonic acid as a substrate and a recombinant aspartate enzyme mutant as a biocatalyst to generate R-3-aminobutyric acid.

[0015] Furthermore, this application uses whole-cell wet bacteria obtained by IPTG induction culture of recombinant engineered bacteria containing the aspartate enzyme mutant encoding gene as the enzyme source, crotonic acid as the substrate, and PBS buffer as the reaction medium to carry out the enzyme catalytic reaction under water bath temperature control and magnetic stirring conditions.

[0016] Further, the wet bacterial enzyme source was prepared as follows: Engineered bacteria containing the relevant gene expression vector were inoculated into 5 mL of LB liquid medium (kanamycin concentration 50 μg / mL) and cultured overnight at 37°C for 12 h to obtain a seed culture. Subsequently, 1 mL of the seed culture was transferred to 500 mL of the same LB liquid medium containing resistance and cultured at 37°C with shaking until OD... 600 When the concentration reaches 0.6–0.8, add IPTG inducer to a final concentration of 0.5 mM. Induce culture at 25°C for 20 h. After culture, centrifuge the bacterial cell culture at 8000 rpm for 20 min, discard the supernatant, and use the collected bacterial cells as an enzyme source, storing them at -80°C for later use.

[0017] The catalytic activities of the single-point mutant aspartate enzymes Asp-L165P (amino acid sequence SEQ ID NO.3) and Asp-M387A (amino acid sequence SEQ ID NO.4) were increased compared with the wild-type enzyme activity (186 U / g), reaching 248 U / g and 293 U / g, respectively; the activity of the two-point combined mutant aspartate enzyme Asp-L165P / M387A (amino acid sequence SEQ ID NO.5) reached 362 U / g. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the reaction of aspartate enzyme in the present invention catalyzing the reaction of crotonic acid to produce R-3-aminobutyric acid;

[0019] Figure 2 Maps of wild-type and mutant aspartate enzyme expression vectors;

[0020] Figure 3 The purity detection spectrum of R-3-aminobutyric acid after extraction and purification catalyzed by the aspartic acid mutant Asp-L165P / M387A;

[0021] Figure 4 Chiral detection spectrum of R-3-aminobutyric acid extracted and purified by the aspartic acid mutant Asp-L165P / M387A. Detailed Implementation

[0022] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0023] Example 1

[0024] I. Construction of wild-type aspartic acid cloning strains

[0025] The amino acid sequence of aspartic acid enzyme from *Pseudomonas flamescens* was obtained from the UniProt protein database. Codon optimization and gene synthesis services were commissioned to Jiangsu Saisofe Biotechnology Co., Ltd., and the gene sequence is shown in SEQ ID NO.1. This gene was cloned into the pET-30a(+) plasmid between the NdeI and NotI restriction sites to obtain the pET30a-Asp recombinant plasmid. The recombinant plasmid pET30a-Asp was heat-shocked into *E. coli* BL21(DE3) competent cells to obtain the wild-type aspartic acid enzyme expression engineered strain BL21-AspWT.

[0026] The engineered strain BL21-AspWT was cultured live on LB medium. LB medium preparation method: 5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl. After preparation, autoclave at 121℃ for 20 min. Separately add 2% (w / v) agar to prepare LB solid medium for plate culture and single colony isolation and purification. Add kanamycin to a final concentration of 50 μg / mL before use. Activation and culture method: Streaking of glycerol tubes containing the engineered strain onto LB solid medium plates for activation, followed by overnight incubation at 37℃. Subsequently, single colonies were picked from the plates and inoculated into 5 mL of LB resistant liquid medium, incubated overnight at 37℃ and 220 rpm. Cells were collected by centrifugation, and plasmids were extracted according to the instructions of the plasmid extraction kit.

[0027] II. Construction of Aspartate Mutant Cloning Strains

[0028] Using the extracted wild-type aspartate expression vector as a DNA template, the aspartate gene was mutated using a PCR mutagenesis kit (MutExpress II Fast Mutagenesis KitV2, Nanjing Novizan Biotechnology Co., Ltd.). The L165P and M387A mutation primers are shown in Table 1, the PCR reaction system composition is shown in Table 2, and the PCR reaction conditions are shown in Table 3.

[0029] Table 1 Primers used for PCR mutation

[0030]

[0031] Table 2 PCR reaction system

[0032]

[0033] Table 3 PCR reaction conditions

[0034]

[0035] After the PCR products were verified to be consistent with the theoretical results by agarose gel electrophoresis, the template plasmid was digested with DpnI restriction endonuclease. The digested product was transformed into E. coli BL21(DE3) competent cells, then plated on LB kanamycin-resistant plates and incubated overnight at 37°C. Single colonies were randomly picked from the plates, liquid cultured, and the plasmid was extracted and sequenced for verification. Finally, the mutant plasmid with the correct sequence was obtained, indicating that the expression vector containing the aspartate aminotransferase mutant gene and the recombinant engineered bacteria were obtained. Finally, the bacterial strain was stored in glycerol tubes at -80°C. The expression vectors of the aspartate aminotransferase mutant gene were named pET30a-Asp-L165P, pET30a-Asp-M387A, and pET30a-Asp-L165P / M387A, respectively. The engineered bacteria were named BL21-Asp-L165P, BL21-Asp-M387A, and BL21-Asp-L165P / M387A, respectively.

[0036] Example 2

[0037] I. Culture of engineered bacteria expressing aspartic acid enzyme

[0038] Engineered bacteria containing the relevant gene were inoculated into 5 mL of LB kanamycin-resistant (50 μg / mL) liquid medium and cultured overnight (12–16 h) at 37°C and 220 rpm. Subsequently, they were transferred to 500 mL of fresh LB liquid medium with the same resistance and cultured at 37°C and 220 rpm with shaking until OD reached. 600 When the concentration reaches 0.6–0.8, IPTG inducer is added to a final concentration of 0.5 mM, and the cells are induced and cultured at 25°C for 20 h. After culture, the fermentation broth is centrifuged at 8000 rpm for 20 min, the supernatant is discarded, and the collected bacterial cells are stored at -80°C for later use. The frozen whole-cell cells are used as the crude enzyme source for enzyme-catalyzed reactions.

[0039] II. Assay of Aspartate Proteinase Activity

[0040] The enzyme activity U of aspartate is defined as the amount of aspartate enzyme required to catalyze the production of 1 micromole of R-3-aminobutyric acid from crotonic acid per minute, which is one enzyme activity unit, i.e., 1U.

[0041] Cell disruption: Take 3g of frozen whole bacterial cells, resuspend them in 15mL PBS buffer (concentration 20mM, pH=8.0), sonicate for 20min, centrifuge at 8000rpm for 10min, and collect the supernatant to obtain crude enzyme solution.

[0042] Enzyme activity assay: 20 mL of reaction solution (pH = 8.0) contained 0.3 M crotonic acid, 5 mM MgSO4, 0.5 M NH4Cl, and 100 mM PBS buffer. Then, 5 mL of crude enzyme solution was added to the reaction solution, and the mixture was incubated at 35 °C and 100 rpm for 20 min with shaking. After the reaction was complete, 1 mL of sample was taken, and 1 mL of acetonitrile was added to terminate the reaction. After centrifugation, the supernatant was derivatized with 2,4-dinitrofluorobenzene and analyzed by HPLC. The enzyme activity was calculated based on the peak area.

[0043] Enzyme activity tests showed that the wild-type enzyme activity was 186 U / g, the single-point mutant Asp-L165P (SEQ ID NO.3) enzyme activity was 248 U / g, the single-point mutant Asp-M387A (SEQ ID NO.4) enzyme activity was 293 U / g, and the two-point combined mutant Asp-L165P / M387A (SEQ ID NO.5) enzyme activity reached 362 U / g.

[0044] Example 3: Genetically engineered bacteria catalyze the production of R-3-aminobutyric acid from crotonic acid.

[0045] Using the engineered bacterium BL21-Asp-L165P constructed in Example 1 as the research object, whole-cell crude enzyme was prepared according to the method in Example 2. 40g of crotonic acid, 0.5g of magnesium sulfate, and 5g of ammonium sulfate were weighed out. The pH was adjusted to 9.0 with ammonia water, and the volume was brought to 200mL with pure water. Stirring was started and the temperature was controlled at 35℃. 10g of crude enzyme was added to start the enzyme-catalyzed reaction. During the reaction, the pH was controlled at 8.5 with ammonia water. After 16 hours of reaction, a sample was taken for HPLC analysis. The conversion rate of R-3-aminobutyric acid in the solution reached 93.7%.

[0046] Example 4: Genetically engineered bacteria catalyze the production of R-3-aminobutyric acid from crotonic acid.

[0047] Using the engineered bacterium BL21-Asp-M387A constructed in Example 1 as the research object, whole-cell crude enzyme was prepared according to the method in Example 2. 40g of crotonic acid, 0.5g of magnesium sulfate, and 5g of ammonium sulfate were weighed out. The pH was adjusted to 9.0 with ammonia water, and the volume was brought to 200mL with pure water. Stirring was started and the temperature was controlled at 35℃. 10g of crude enzyme was added to start the enzyme-catalyzed reaction. During the reaction, the pH was controlled at 8.5 with ammonia water. After 16 hours of reaction, a sample was taken for HPLC analysis. The conversion rate of R-3-aminobutyric acid in the solution reached 94.6%.

[0048] Example 5: Genetically engineered bacteria catalyze the production of R-3-aminobutyric acid from crotonic acid.

[0049] Using the engineered bacterium BL21-Asp-L165P / M387A constructed in Example 1 as the research object, whole-cell crude enzyme was prepared according to the method in Example 2. 40g of crotonic acid, 0.5g of magnesium sulfate, and 5g of ammonium sulfate were weighed according to the example. The pH was adjusted to 9.0 with ammonia water, and the volume was brought to 200mL with pure water. Stirring was started and the temperature was controlled at 35℃. 10g of crude enzyme was added to start the enzyme-catalyzed reaction. During the reaction, the pH was controlled at 8.5 with ammonia water. After 16 hours of reaction, a sample was taken for HPLC analysis. The conversion rate of R-3-aminobutyric acid in the solution reached 95.6%.

[0050] Example 6: Optimization of the enzyme catalytic system for the production of R-3-aminobutyric acid from crotonic acid.

[0051] I. Optimization of substrate addition in enzyme-catalyzed reaction system

[0052] Using the engineered bacterium BL21-Asp-L165P / M387A constructed in Example 1 as the research object, whole-cell crude enzyme was prepared using the method in Example 2. The enzyme-catalyzed reaction system was configured according to Example 3, with three gradients of crotonic acid addition: 40g, 60g, and 80g. The pH was then adjusted to 9.0 with ammonia, and the volume was brought to 200mL with pure water. Stirring was started and the temperature was controlled at 35℃. After the reaction solution was prepared, 10g of crude enzyme was added to each reaction to begin the enzyme-catalyzed reaction. The pH was maintained at 8.5 with ammonia during the reaction. After 16 hours of reaction, samples were taken for HPLC analysis. The results showed that the conversion rates were 98.2%, 97.9%, and 93.4% when crotonic acid was added at concentrations of 40g, 60g, and 80g, respectively.

[0053] II. Optimization of Enzyme Dosage in Enzyme Catalytic Reaction Systems

[0054] Using the engineered bacterium BL21-Asp-L165P / M387A constructed in Example 1 as the research object, whole-cell crude enzyme was prepared using the method in Example 2. The enzyme-catalyzed reaction system was prepared according to Example 3, with 60g of crotonic acid added. Then, the pH was adjusted to 9.0 with ammonia, and the volume was brought to 200mL with pure water. Stirring was started and the temperature was controlled at 35℃. After the reaction solution was prepared, the crude enzyme addition amounts were set to 10g, 8g, and 5g, respectively. The enzyme-catalyzed reaction was started, and the pH was controlled at 8.5 with ammonia during the reaction. After 16 hours of reaction, samples were taken for HPLC analysis. The results showed that the conversion rates were 97.6%, 97.3%, 97.4%, and 93.6% when the crude enzyme addition amounts were 10g, 8g, 5g, and 2.5g, respectively.

[0055] III. Optimization of pH and Temperature in Enzyme Catalytic Reaction Systems

[0056] Using the engineered bacterium BL21-Asp-L165P / M387A constructed in Example 1 as the research object, whole-cell crude enzyme was prepared using the method in Example 2. 60g of crotonic acid, 0.5g of magnesium sulfate, and 5g of ammonium sulfate were weighed according to the example. The pH was adjusted to 9.0 with ammonia water, and the volume was brought to 200mL with pure water. Stirring was started and the temperature was controlled at the predetermined value. 5g of crude enzyme was added to start the enzyme-catalyzed reaction. During the reaction, the pH was controlled at the predetermined value with ammonia water. After 16 hours of reaction, samples were taken for HPLC analysis. By adjusting the temperature and pH, the optimal pH and temperature for enzyme catalysis were determined (Table 4).

[0057] Table 4. Optimization of pH and temperature for enzyme-catalyzed reactions

[0058]

[0059] In summary, by optimizing the enzyme-catalyzed reaction conditions, the conversion rate of R-3-aminobutyric acid can reach over 99%, which meets the requirements of large-scale industrial production.

[0060] Example 7: Small-scale enzyme-catalyzed reaction

[0061] Based on the results obtained in Example 6, a small-scale 2L enzyme-catalyzed reaction was conducted. In the 2L catalytic system, 600g of crotonic acid, 5g of magnesium sulfate, and 50g of ammonium sulfate were added. The pH was adjusted to 9.0 with ammonia water, and the mixture was stirred at 40°C. Then, 50g of the aspartate aminotransferase mutant Asp-L165P / M387A whole-cell crude enzyme was added to initiate the reaction. After 16 hours of reaction, the conversion rate of R-3-aminobutyric acid in the solution was measured to be 99.8%.

[0062] After the enzyme-catalyzed reaction, the reaction solution was passed through a ceramic filtration membrane and an ultrafiltration membrane to remove bacterial cell debris and protein impurities, yielding a filtrate. The filtrate was further decolorized with activated carbon, concentrated under vacuum, and then 2-5 times its volume of ethanol solution was added to the concentrate for low-temperature crystallization. After crystals precipitated, the solution was filtered and dried to obtain pure R-3-aminobutyric acid. Testing showed that the R-3-aminobutyric acid yield was over 85%, the purity was >99%, and the ee value was >99.9%.

[0063] SEQ ID NO.1

[0064]

[0065] SEQ ID NO.2

[0066]

[0067]

[0068] SEQ ID NO.3

[0069]

[0070]

[0071] SEQ ID NO.4

[0072]

[0073]

[0074] SEQ ID NO.5

[0075]

[0076] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. For those skilled in the art, various improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An aspartate enzyme mutant, characterized in that: The mutant was obtained by single or double mutation of leucine at position 165 and / or methionine at position 387 in the amino acid sequence of wild-type aspartic acidase from Pseudomonas flamescens as shown in SEQ ID NO.2; wherein leucine at position 165 was mutated to proline and methionine at position 387 was mutated to alanine.

2. The aspartate enzyme mutant according to claim 1, characterized in that: The mutant is formed by mutating leucine at position 165 of the amino acid sequence shown in SEQ ID NO.2 to proline, and the mutant aspartate enzyme sequence is shown in SEQ ID NO.

3.

3. The aspartate enzyme mutant according to claim 1, characterized in that: The mutant is formed by mutating methionine at position 387 of the amino acid sequence shown in SEQ ID NO.2 to alanine, and the mutant aspartate enzyme sequence is shown in SEQ ID NO.

4.

4. The aspartate enzyme mutant according to claim 1, characterized in that: The mutant is obtained by mutating leucine at position 165 of the amino acid sequence shown in SEQ ID NO.2 to proline, and methionine at position 387 to alanine, resulting in a two-point combination mutant. The mutant aspartate enzyme sequence is shown in SEQ ID NO.

5.

5. A recombinant expression vector constructed from the aspartic acid mutant sequence of any one of claims 1-4.

6. A recombinant genetically engineered bacterium prepared by transformation of the recombinant expression vector according to claim 5.

7. The use of the aspartic acid enzyme mutant as described in any one of claims 1-4 in the catalytic preparation of R-3-aminobutyric acid.

8. The application in the catalytic preparation of R-3-aminobutyric acid according to claim 7, characterized in that: Using crotonic acid as a substrate and a recombinant aspartate enzyme mutant as a biocatalyst, R-3-aminobutyric acid was generated through the reaction.

9. The application in the catalytic preparation of R-3-aminobutyric acid according to claim 8, characterized in that: The enzyme catalysis was carried out using whole-cell wet bacteria obtained by IPTG induction culture of recombinant engineered bacteria containing the aspartate mutant encoding gene as the enzyme source, crotonic acid as the substrate and PBS buffer as the reaction medium, under water bath temperature control and magnetic stirring conditions.

10. The application in the catalytic preparation of R-3-aminobutyric acid according to claim 9, characterized in that: The method for preparing the wet bacterial enzyme source is as follows: inoculate the engineered bacteria containing the relevant gene expression vector into 5 mL of LB liquid medium, and culture overnight at 37°C for 12 h to obtain the seed liquid; Subsequently, 1 mL of seed culture was transferred to 500 mL of the same resistant LB liquid medium and cultured at 37°C with shaking until the OD600 reached 0.6–0.

8. IPTG inducer was then added to a final concentration of 0.5 mM. The culture was induced at 25°C for 20 h. After the culture was completed, the bacterial cell culture was centrifuged at 8000 rpm for 20 min, the supernatant was discarded, and the collected bacterial cells were used as the enzyme source and stored at -80°C for later use.

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