Enzymatic preparation process of R-3-aminobutyric acid

Abstract

The invention provides an enzymatic preparation method of R-3-aminobutyric acid, and belongs to the technical field of biochemistry. According to the method, crotonic acid is taken as a substrate, a recombinant aspartase mutant is taken as a biocatalyst, an enzyme catalysis reaction is carried out at a proper temperature and under pH regulation, and the product R-3-aminobutyric acid is prepared with high efficiency and high stereoselectivity. According to the method, the recombinant aspartase is subjected to heterologous expression by taking escherichia coli as a host. Expression host whole cells are used as crude enzyme for enzyme catalysis reaction, reaction liquid is extracted and purified to obtain a high-purity R-3-aminobutyric acid product, the conversion rate reaches 99% or above, and the product purity is larger than 99% and larger than 99.9% ee. The method provided by the invention has the characteristics of high conversion rate and high yield, and is easy to industrialize.

Description

Technical Field

[0001] The invention relates to an enzymatic preparation process of R-3-aminobutyric acid, belonging to the technical field of biochemistry. Technical Background

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

[0003] At present, the methods for preparing R-3-aminobutyric acid mainly include chemical synthesis and biological methods. Among them, the chemical synthesis method has complicated processes and high production costs, limited product stereoselectivity, and the reaction process involves high temperature and high pressure operations, which causes great pollution to the environment, which is not conducive to industrial scale-up production. Therefore, the preparation of R-3-aminobutyric acid by bioenzymatic method has gradually become the focus of industrialization.

[0004] The bioenzymatic method has the advantages of strong substrate specificity, mild reaction conditions, and strong stereoselectivity, but it has long been limited by the selection of enzyme sequences, making it difficult to improve the substrate conversion rate. Aspartase, also known as aspartate ammonia lyase (Asp, EC4.3.1.1), is one of the three members of the ammonia lyase family. It catalyzes the reversible reaction of L-aspartate to produce ammonia and fumarate, and is of great significance in microbial nitrogen metabolism. It is also an industrial precursor for the synthesis of food additives and artificial sweeteners. The sources of aspartase 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 through enzyme engineering to adapt to crotonic acid. Among them, the mutant enzyme BSASP-C6 catalyzes the preparation of R-3-aminobutyric acid from crotonic acid and ammonia, with a substrate concentration of 300mM and a final conversion rate of about 60% in 100 hours. The conversion time is long and the conversion rate is low, which is not suitable for industrial production.

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

[0006] In order to overcome the above technical defects, the purpose of the present invention is to provide an amino acid sequence of aspartase, and at the same time provide a process for preparing R-3-aminobutyric acid using the enzyme, so as to further promote the industrial production of R-3-aminobutyric acid. With crotonic acid as a substrate and a recombinant aspartase mutant as a biocatalyst, an enzyme catalytic reaction is carried out under appropriate temperature and pH control to prepare the product R-3-aminobutyric acid efficiently and with high stereoselectivity. The method uses Escherichia coli as a host to heterologously express the recombinant aspartase. The whole cell of the expression host is used as a crude enzyme for the enzyme catalytic reaction, and the reaction solution is extracted and purified to obtain a high-purity R-3-aminobutyric acid product, with a conversion rate of more than 99%, a product purity of >99%, and >99.9%ee. The method of the present invention has the characteristics of high conversion rate and high yield, and is easy to industrialize.

[0007] The aspartase mutant of the present invention is obtained by subjecting the amino acid sequence of the wild-type aspartase from Pseudomonas flavescens (shown in SEQ ID NO.2) to single mutation or double mutation of the 165th leucine or the 387th methionine.

[0008] Furthermore, the mutant is a mutant in which the leucine at position 165 of the amino acid sequence shown in SEQ ID NO.2 is mutated to proline, and the mutant aspartase sequence is shown in SEQ ID NO.3.

[0009] Furthermore, the mutant is a mutant in which the methionine at position 387 of the amino acid sequence shown in SEQ ID NO.2 is mutated to alanine, and the mutant aspartase sequence is shown in SEQ ID NO.4.

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

[0011] The present invention also provides a recombinant expression vector constructed by the aforementioned aspartase mutant sequence.

[0012] The present invention also provides a recombinant genetic engineering bacterium prepared by transformation of the above-mentioned recombinant expression vector.

[0013] In addition, the present invention also provides an application of utilizing the aspartase mutant to catalyze the preparation of R-3-aminobutyric acid from crotonic acid.

[0014] Furthermore, the application uses crotonic acid as a substrate and a recombinant aspartase mutant as a biocatalyst to react and generate R-3-aminobutyric acid.

[0015] Furthermore, the application uses whole-cell wet bacteria obtained by IPTG-induced culture of recombinant engineered bacteria containing an aspartase mutant encoding gene as an enzyme source, crotonic acid as a substrate, and PBS buffer as a reaction medium, and carries out enzyme-catalyzed reaction under water bath temperature-controlled magnetic stirring conditions.

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

[0017] The catalytic activities of the single-point mutant aspartase Asp-L165P (amino acid sequence is SEQ ID NO.3) and Asp-M387A (amino acid sequence is SEQ ID NO.4) were both improved compared with the wild-type enzyme activity (186U / g), reaching 248U / g and 293U / g respectively; the enzyme activity of the two-point combined mutation aspartase Asp-L165P / M387A (amino acid sequence is SEQ ID NO.5) reached 362U / g. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic diagram of the reaction of aspartase of the present invention catalyzing the reaction of crotonic acid to generate R-3-aminobutyric acid;

[0019] Figure 2 The following is the map of the expression vectors of wild-type and mutant aspartase;

[0020] Figure 3 This is the purity detection chart of the synthesis of R-3-aminobutyric acid catalyzed by the aspartase mutant Asp-L165P / M387A after extraction and purification;

[0021] Figure 4 This is the chiral detection spectrum of the synthesis of R-3-aminobutyric acid catalyzed by the aspartase mutant Asp-L165P / M387A after extraction and purification. DETAILED DESCRIPTION

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

[0023] Example 1

[0024] 1. Construction of wild-type aspartase cloning strain

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

[0026] The engineered bacteria BL21-AspWT was cultured live in LB medium. LB medium preparation method: 5g / L yeast powder, 10g / L peptone, 10g / L NaCl. After preparation, sterilize with high pressure steam at 121℃ for 20min for standby use. Add 2% (w / v) agar as LB solid medium for strain plate culture and single colony isolation and purification. Add kanamycin at a final concentration of 50μg / mL before using the medium. Activation and culture method: The glycerol tube containing the engineered bacteria was streaked on the LB solid medium plate for separation and activation, and cultured at 37℃ overnight. Subsequently, a single colony was picked from the plate and inoculated into 5mL LB resistance liquid medium, and cultured overnight at 37℃ and 220rpm. The bacterial cells were collected by centrifugation, and plasmid extraction was performed according to the operating instructions of the plasmid extraction kit.

[0027] 2. Construction of aspartase mutant cloning strain

[0028] The wild-type aspartase expression vector extracted above was used as a DNA template, and a PCR mutation kit (MutExpress II Fast Mutagenesis Kit V2, Nanjing Novogene Biotech Co., Ltd.) was used to perform gene mutation on aspartase. 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 product was verified by agarose gel electrophoresis to be consistent with the theory, the template plasmid was digested with DpnI restriction endonuclease. The digested product was transformed into Escherichia coli BL21 (DE3) competent cells, then coated on LB kanamycin resistance plates and cultured overnight at 37°C. Single colonies on the plate were randomly picked, and the plasmids were extracted and sequenced after liquid culture. Finally, a mutant plasmid with the correct sequence was obtained, indicating that an expression vector and recombinant engineered bacteria containing the aspartase mutant gene were obtained. Finally, the strains were stored in a glycerol tube in a -80°C refrigerator. The expression vectors of the aspartase 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] 1. Cultivation of engineered bacteria expressing aspartase

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

[0039] 2. Determination of Aspartase Activity

[0040] The enzyme activity U of aspartase is defined as: the amount of aspartase used to catalyze crotonic acid to produce 1 micromole of R-3-aminobutyric acid per minute is one enzyme activity unit, i.e. 1U.

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

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

[0043] According to enzyme activity detection, the wild-type enzyme activity is 186U / g, the single-point mutation Asp-L165P (SEQ ID NO.3) has an enzyme activity of 248U / g, the single-point mutation Asp-M387A (SEQ ID NO.4) has an enzyme activity of 293U / g, and the two-point combination mutation Asp-L165P / M387A (SEQ ID NO.5) has an enzyme activity of 362U / g.

[0044] Example 3 Genetically engineered bacteria catalyze crotonic acid to produce R-3-aminobutyric acid

[0045] The engineered bacteria BL21-Asp-L165P constructed in Example 1 was used as the research object, and the whole cell crude enzyme was prepared by the method in Example 2. Weigh 40g of crotonic acid, 0.5g of magnesium sulfate, and 5g of ammonium sulfate respectively, adjust the pH to 9.0 with ammonia water, dilute to 200mL with pure water, start stirring and control the temperature to 35°C. Add 10g of crude enzyme, start the enzyme catalytic reaction, control the pH to 8.5 with ammonia water during the reaction, and after 16h of reaction, take a sample for HPLC detection. The conversion rate of R-3-aminobutyric acid in the detected solution reached 93.7%.

[0046] Example 4 Genetically engineered bacteria catalyze crotonic acid to produce R-3-aminobutyric acid

[0047] The engineered bacteria BL21-Asp-M387A constructed in Example 1 was used as the research object, and the whole cell crude enzyme was prepared by the method in Example 2. Weigh 40g of crotonic acid, 0.5g of magnesium sulfate, and 5g of ammonium sulfate respectively, adjust the pH to 9.0 with ammonia water, set the volume to 200mL with pure water, start stirring and control the temperature to 35°C. Add 10g of crude enzyme, start the enzyme catalytic reaction, control the pH to 8.5 with ammonia water during the reaction, and after 16h of reaction, take samples for HPLC detection. The conversion rate of R-3-aminobutyric acid in the detected solution reached 94.6%.

[0048] Example 5 Genetically engineered bacteria catalyze crotonic acid to produce R-3-aminobutyric acid

[0049] The engineering bacteria BL21-Asp-L165P / M387A constructed in Example 1 was used as the research object, and the whole cell crude enzyme was prepared by the method in Example 2. According to the example, 40g of crotonic acid, 0.5g of magnesium sulfate, and 5g of ammonium sulfate were weighed respectively, the pH was adjusted to 9.0 with ammonia water, the volume was made up to 200mL with pure water, stirring was turned on and the temperature was controlled at 35°C. 10g of crude enzyme was added, and the enzyme catalytic reaction was started. During the reaction, the pH was controlled at 8.5 with ammonia water. After 16h of reaction, sampling was performed for HPLC detection. The conversion rate of R-3-aminobutyric acid in the detected solution reached 95.6%.

[0050] Example 6 Optimization of the enzyme catalytic system for catalyzing crotonic acid to produce R-3-aminobutyric acid

[0051] 1. Optimization of substrate addition amount in enzyme-catalyzed reaction system

[0052] The engineered bacteria BL21-Asp-L165P / M387A constructed in Example 1 was used as the research object, and the whole cell crude enzyme was prepared by the method in Example 2. The enzyme catalytic reaction system was configured according to Example 3, wherein the amount of crotonic acid added was set to 3 gradients, 40g, 60g, and 80g, respectively. Then, the pH was adjusted to 9.0 with ammonia water, the volume was fixed to 200mL with pure water, stirring was turned on and the temperature was controlled at 35°C. After the reaction solution was configured, 10g of crude enzyme was added to start the enzyme catalytic reaction, and the pH was controlled at 8.5 with ammonia water during the reaction. After 16h of reaction, sampling was performed for HPLC detection. After testing, the conversion rates were 98.2%, 97.9%, and 93.4% when the amount of crotonic acid added was 40g, 60g, and 80g, respectively.

[0053] 2. Optimization of enzyme dosage in enzyme-catalyzed reaction system

[0054] The engineered bacteria BL21-Asp-L165P / M387A constructed in Example 1 was used as the research object, and the whole-cell crude enzyme was prepared by the method in Example 2. The enzyme catalytic reaction system was configured according to Example 3, wherein the amount of crotonic acid added was 60g. Then, the pH was adjusted to 9.0 with ammonia water, the volume was fixed to 200mL with pure water, stirring was turned on and the temperature was controlled at 35°C. After the reaction solution was configured, the amount of crude enzyme added was set to 10g, 8g, and 5g, respectively. The enzyme catalytic reaction was started, and the pH was controlled to 8.5 with ammonia water during the reaction. After 16h of reaction, sampling was performed for HPLC detection. After testing, 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] 3. Optimization of pH and temperature of enzyme-catalyzed reaction system

[0056] With the engineering bacteria BL21-Asp-L165P / M387A constructed in Example 1 as research object, the whole cell crude enzyme was prepared by the method in Example 2. According to the example, 60g crotonic acid, 0.5g magnesium sulfate, 5g ammonium sulfate were weighed respectively, pH was adjusted to 9.0 with ammonia water, and the volume was adjusted to 200mL with pure water, and stirring was turned on and the temperature was controlled at a predetermined value. 5g of crude enzyme was added, and enzyme catalysis reaction was started. During the reaction, pH was controlled at a predetermined value with ammonia water, and after 16h of question reaction, HPLC detection was performed by sampling. By adjusting temperature and pH, the optimum pH and temperature (Table 4) for enzyme catalysis were detected.

[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 more than 99%, which meets the needs of large-scale industrial production.

[0060] Example 7 Enzyme catalytic reaction test

[0061] According to the results obtained in Example 6, a 2L enzyme catalytic reaction test was carried out. In the 2L catalytic system, 600g of crotonic acid, 5g of magnesium sulfate, and 50g of ammonium sulfate were added, and the pH was adjusted to 9.0 with ammonia water, stirred at 40°C, and 50g of whole cell crude enzyme of aspartase mutant Asp-L165P / M387A was added to start the reaction. After the reaction was completed for 16h, the conversion rate of R-3-aminobutyric acid in the solution was detected to reach 99.8%.

[0062] After the enzyme catalytic reaction is completed, the reaction solution is filtered through a ceramic filter membrane and an ultrafiltration membrane to remove impurities such as bacterial cell fragments and proteins to obtain a filtrate. The filtrate is further decolorized with activated carbon, and the liquid is concentrated by vacuum decompression. Subsequently, 2-5 times the volume of ethanol solution is added to the concentrate for low-temperature crystallization. After the crystals are precipitated, they are filtered and dried to obtain pure R-3-aminobutyric acid. According to testing, the yield of R-3-aminobutyric acid product is more than 85%, the product purity is greater than 99%, and the ee value is greater than 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 is only a preferred embodiment of the present invention, but the protection scope of the present invention is not limited thereto. For those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered as the protection scope of the present invention.

Claims

1. An aspartase mutant, characterized in that: The mutant is obtained by subjecting the amino acid sequence of wild-type aspartase from Pseudomonas flavescens (shown in SEQ ID NO.2) to single mutation or double mutation of the 165th leucine and / or the 387th methionine.

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

3.

3. The aspartase mutant according to claim 1, characterized in that: The mutant is obtained by mutating the 387th methionine in the amino acid sequence shown in SEQ ID NO.2 to alanine, and the mutant aspartase sequence is shown in SEQ ID NO.

4.

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

5.

5. A recombinant expression vector constructed using the aspartase mutant sequence according to any one of claims 1 to 4.

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

7. Use of the aspartase mutant according to any one of claims 1 to 4 in catalyzing the preparation of R-3-aminobutyric acid.

8. The use of the method according to claim 7 in the catalytic preparation of R-3-aminobutyric acid, characterized in that: Using crotonate as substrate and recombinant aspartase mutant as biocatalyst, the reaction produces R-3-aminobutyric acid.

9. The use of the catalytic preparation of R-3-aminobutyric acid according to claim 8, characterized in that: The aspartase mutant is used to catalyze the enzyme reaction, with the whole-cell wet bacteria obtained by IPTG induction culture of the recombinant engineering bacteria containing the aspartase mutant encoding gene as the enzyme source, crotonic acid as the substrate and PBS buffer as the reaction medium, under the condition of water bath temperature control and magnetic stirring.

10. The use of the catalyst in the preparation of R-3-aminobutyric acid according to claim 9, characterized in that: The wet bacterial enzyme source preparation method is to inoculate the engineering bacteria containing the relevant gene expression vector into 5 mL LB liquid culture medium, culture overnight at 37° C. for 12 hours, and obtain seed liquid; Then, transfer 1 mL of seed solution to 500 mL of LB liquid medium containing the same resistance, and culture at 37°C with shaking until OD 600 When the pH value reaches 0.6-0.8, add IPTG inducer to a final concentration of 0.5 mM; induce and culture at 25°C for 20 h; after the culture, centrifuge the bacterial cell culture at 8000 rpm for 20 min, discard the supernatant, and store the collected bacterial cells as an enzyme source at -80°C for later use.

Images