Method for biologically synthesizing succinic acid in vitro by taking L-glutamic acid as raw material

Abstract

The invention relates to the technical field of enzyme catalysis, and particularly discloses a method for biologically synthesizing succinic acid in vitro by taking L-glutamic acid as a raw material, which constructs a new path for synthesizing L-glutamic acid-3-cyanopropionic acid-succinic acid by a full-biological enzyme method for the first time. A green conversion chain from cheap and bulk amino acids to a high value-added biodegradable plastic precursor is broken through, an L-glutamic acid resource with leading productivity is in butt joint with a poly (butylene succinate) (PBS) industry chain in shortage in the market, and the whole process of finally synthesizing succinic acid from L-glutamic acid through a 3-cyanopropionic acid intermediate is realized. According to the process, full-water-phase and extremely-mild one-pot method or multi-enzyme coupling conversion is realized. The two-step reaction is carried out in a nearly neutral water phase system, and the compatibility of enzyme is excellent, so that the process flow is greatly simplified, the equipment investment and energy consumption are reduced, common harmful byproducts in chemical synthesis are reduced from the source, and the biological safety and extremely high purity of the product succinic acid are ensured.

Description

Technical Field

[0001] This invention relates to the field of enzyme catalysis technology, and specifically discloses a method for the in vitro biosynthesis of succinic acid using L-glutamic acid as a raw material. Background Technology

[0002] Succinic acid is a key precursor to biodegradable plastic PBS. Developing green synthesis technology for succinic acid with independent intellectual property rights is of great significance for ensuring the safety of the industrial chain.

[0003] According to the China Plastics Processing Industry Association, the market size of biodegradable plastics in my country is projected to exceed 50 billion yuan by 2030. Among them, polybutylene succinate (PBS), currently recognized worldwide as the best-performing biodegradable plastic (completely biodegradable into carbon dioxide and water), will account for more than 30% of the market share. As a key precursor in PBS synthesis (… Figure 1 The green and efficient production technology of succinic acid is directly related to the independent control of the entire biodegradable plastics industry chain. However, at present, my country's succinic acid production still mainly relies on the petroleum-based maleic anhydride hydrogenation process, which has prominent problems such as raw material dependence on the international crude oil market and high energy consumption and emissions during the production process. In July 2024, the Ministry of Industry and Information Technology and other departments issued the "Implementation Plan for the Innovative Development of the Fine Chemical Industry (2024-2027)" (MIIT Joint Document

[2024] No. 136), proposing to focus on the development of bio-based polymers such as lactic acid, 1,3-propanediol, acrylic acid, and succinic acid to effectively supplement existing fossil-based materials. Therefore, developing a new environmentally friendly, efficient, and low-carbon biosynthesis process for succinic acid is of great significance for breaking the foreign technology monopoly and ensuring the security of the industry chain. Summary of the Invention

[0004] In view of the prior art, the first aspect of the present invention provides a method for in vitro biosynthesis of succinic acid, comprising the steps of: 3-Cyanopyranoic acid is mixed with the first enzyme in the first buffer solution to obtain the first mixture, and the reaction yields succinic acid. .

[0005] The 3-cyanopropionic acid is synthesized from L-glutamic acid in one or more steps.

[0006] In some specific embodiments of the method for in vitro biosynthesis of succinic acid described in the first aspect, the first enzyme is selected from proteins that have more than 90% homology with the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 and have nitrile hydrolase function.

[0007] In some specific embodiments of the method for in vitro biosynthesis of succinic acid described in the first aspect, each 1L of the first buffer solution includes 40-60 mmol of 3-cyanopropionic acid.

[0008] In some specific embodiments of the method for in vitro biosynthesis of succinic acid described in the first aspect, each 1L of the first buffer solution contains 80-120mmol of the first enzyme.

[0009] In some specific embodiments of the method for in vitro biosynthesis of succinic acid described in the first aspect, the first buffer solution is PB buffer solution.

[0010] In some specific embodiments of the method for in vitro biosynthesis of succinic acid described in the first aspect, the reaction temperature is 25-37°C.

[0011] In some specific embodiments of the in vitro biosynthesis of succinic acid described in the first aspect, the method for synthesizing the substrate 3-cyanopropionic acid is as follows: L-glutamic acid, H2O2, haloperoxidase VHPOs, second buffer solution, and NaBr were mixed to obtain a second mixture, and the reaction yielded 3-cyanopropionic acid. .

[0012] In some specific embodiments of the method for in vitro biosynthesis of succinic acid described in the first aspect, the halogen peroxidase VHPOs are selected from proteins having more than 90% homology with the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

[0013] In some specific embodiments of the method for in vitro biosynthesis of succinic acid described in the first aspect, each 1L of the second mixture includes 8.0~12.0mmol of L-glutamic acid.

[0014] In some specific embodiments of the method for in vitro biosynthesis of succinic acid described in the first aspect, each 1L of the second mixture includes 0.4~0.6mol of H2O2.

[0015] In some specific embodiments of the method for in vitro biosynthesis of succinic acid described in the first aspect, the second buffer is a sodium citrate buffer, and the pH value of the second buffer is 5-7.

[0016] In some specific embodiments of the method for in vitro biosynthesis of succinic acid described in the first aspect, each 1L of the second buffer solution comprises 0.4~0.6mmol.

[0017] Advantages of the present invention This invention, for the first time, constructs a novel all-bioenzymatic synthesis pathway using L-glutamic acid—3-cyanopropionic acid—succinic acid, opening up a green conversion chain from inexpensive, bulk amino acids to high-value-added biodegradable plastic precursors. It connects the leading-capacity L-glutamic acid resources with the market-scarce polybutylene succinate (PBS) industry chain, realizing the entire process of synthesizing succinic acid from L-glutamic acid via the 3-cyanopropionic acid intermediate. This process achieves a fully aqueous, extremely mild "one-pot" or multi-enzyme coupled conversion. Based on the novel enzymatic catalytic pathway discovered in this invention, the entire synthesis process is completely free from the reagents required by traditional chemical methods. Since both reactions are carried out in a near-neutral aqueous system, the enzyme compatibility is excellent, greatly simplifying the process flow, reducing equipment investment and energy consumption, and reducing harmful byproducts commonly found in chemical synthesis from the source, ensuring the biosafety and extremely high purity of the succinic acid product. Attached Figure Description

[0018] Figure 1 Application of succinic acid as a key precursor for the biodegradable plastic polybutylene succinate; Figure 2 The route for preparing succinic acid from biomass glutamic acid proposed in this work; Figure 3 Electrophoretic analysis results of SlVCPO haloperoxidase, BvVCPO haloperoxidase, and SjVBPO haloperoxidase; Figure 4 Electrophoretic analysis results of nitrile hydrolase RrNI, nitrile hydrolase GsNI, and nitrile hydrolase GnNI. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1.

[0021] The three target amino acid sequences of the haloperoxidase VHPOs are as follows: Sl The VCPO haloperoxidase sequence is shown in SEQ ID NO:1, source: Stemphylium lycopersici; The sequence of BvVCPO haloperoxidase is as shown in SEQ ID NO:2, and it originates from Bipolaris victoriae FI3; SjThe VBPO haloperoxidase sequence is shown in SEQ ID NO:3, source: Saccharina japonica; The three target amino acid sequences obtained from the screening were submitted to Changzhou Xinyisheng Life Technology Co., Ltd., who were entrusted to optimize the codons according to the codon preference of E. coli. The gene was cloned into the NdeI / XhoI site of the pET28a+ vector using high-throughput synthesis technology, double enzyme digestion and ligation technology, and plasmid construction was carried out. The optimized gene sequence was cloned into the vector (pET28a+ plasmid) and sequenced to ensure the accuracy of the sequence. The constructed plasmid will be used for subsequent heterologous expression and enzyme activity identification experiments.

[0022] Plasmid transformation, protein expression and purification and preparation of crude enzyme solution Plasmid transformation: 2 μL of the constructed plasmid was added to 100 μL of E. coli BL21(DE3) competent cells on a clean bench. After incubation on ice for 30 minutes, the cells were heat-shocked in a 42°C water bath for 45 seconds, followed by incubation on ice for 2 minutes. 700 μL of LB liquid medium was added, and the cells were incubated on a shaker at 37°C and 200 rpm for 1 hour. The mixture was then spread onto LB solid medium containing 50 μg / mL kanamycin sulfate and cultured at 37°C for 12 hours.

[0023] Protein expression: Single colonies were selected and cultured in 5 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate at 37°C on a shaker at 200 rpm until turbidity was observed. The culture was then transferred to a large-volume medium at a 1% inoculum size and cultured under the same conditions. When the OD600 reached 1.1–1.5, 0.5 mM IPTG was added, and the cells were induced at 18°C ​​and 180 rpm for 16 hours. After centrifugation at 12000 rpm and 4°C for 20 min, the supernatant was discarded, and the cells were washed with 50 mM Tirs-H2SO4 (pH=8.2) buffer and centrifuged again to obtain whole bacterial cells, which were then stored at -80°C.

[0024] Protein purification: The collected whole bacterial cells were added to 50 mM Tirs-H2SO4 (pH=8.2) containing 25 mM imidazole. After sonication in an ice-water bath for one hour, the cells were centrifuged at 12,000 rpm for 20 min at 4°C. The supernatant was collected using a syringe and filtered through a 0.22 μm filter. The supernatant was stored on ice. The preservation solution in the nickel column was replaced with ultrapure water. The nickel column was equilibrated with 50 mM Tirs-H2SO4 (pH=8.2) containing 25 mM imidazole. The sample was slowly loaded onto the column using a dropper and allowed to stand on ice for 0.5 h to allow the protein to fully bind to the nickel column. Impurities were eluted with 50 mM Tirs-H2SO4 (pH=8.2) containing 50 mM imidazole, and the target protein was eluted with 50 mM Tirs-H2SO4 (pH=8.2) containing 250 mM imidazole. The eluent was collected in separate tubes and labeled. The protein eluent was transferred to an ultrafiltration tube and ultrafiltered at 4°C and 3900 rpm using a horizontal centrifuge to desalt and concentrate the protein sample. After ultrafiltration, glycerol to a final concentration of 20% and 100 μM Na₂VO₄ were added to the protein sample (to provide the VO₄ required for the reorganization of the active site of the purified enzyme). 3- Protein concentration was determined using the Coomassie Brilliant Blue method, and the samples were flash-frozen in liquid nitrogen and stored at -80°C.

[0025] Preparation of crude enzyme solution: Thaw approximately 1g of wet cells and resuspend them in 50mM Tris / H2SO4 buffer (pH=8.2) (0.1g / mL). After sonication in an ice-water bath for one hour, centrifuge at 12000 rpm for 20 min at 4°C. Collect the supernatant using a syringe and filter through a 0.22μm filter. Store on ice. Incubate the supernatant at 70°C for 2 × 20 min (calculated after the solution reaches 70°C). Centrifuge (10000 rpm, 10 min) after each heat treatment to remove denatured proteins. Using an ultrafiltration tube with a molecular weight cutoff of 30 kDa, exchange the buffer with Tris / H2SO4 buffer (50mM, pH=8.2) four times. Desalt and concentrate the crude enzyme by ultrafiltration using a horizontal centrifuge at 3900 rpm at 4°C. After ultrafiltration, the protein sample was supplemented with glycerol to a final concentration of 20% and 100 μM Na3VO4 (to provide the purified enzyme with the VO4 required for recombination of the active site). 3- Protein concentration was determined using the Coomassie Brilliant Blue method and stored at -80°C.

[0026] The above methods were used to express and purify or prepare crude enzymes of three vanadium-dependent haloperoxidases, which were then analyzed by SDS-PAGE electrophoresis.

[0027] like Figure 3As shown, the SDS-PAGE electrophoresis results showed that the protein samples all had obvious characteristic bands at the corresponding positions, the apparent molecular weight of each target protein was consistent with the expectation, and the target protein bands in each lane were clear and thick, proving that this embodiment successfully constructed and efficiently expressed three vanadium-dependent haloperoxidases with high purity.

[0028]

[0029] The total reaction volume was 2 mL, and the system was as follows: L-glutamic acid (10 mM), H2O2 (0.5 M), enzyme solution ( Sl VCPO haloperoxidase, Bv VCPO haloperoxidase, Sj VBPO haloperoxidase (any one, 100 nM), sodium citrate buffer (pH = 5.6), and NaBr (0.5 mM) were used, with concentrations representing the final concentrations of each reactant. The reaction mixture was stirred on a magnetic stirrer (RT, 1400 rpm). H2O2 was added to the reaction solution using a micro-injection pump at a rate of 0.15 mL / h for 5 hours. After the addition was completed, the reaction was extended to 24 hours. The yield results are shown in Table 1.

[0030] Table 1

[0031] The yield of 3-cyanopropionic acid was analyzed by high performance liquid chromatography (HPLC). The detection conditions were as follows: Horizon AQUA C18 5u HPLC column (catalog number: AQUA-250-46-50), temperature: 28℃, flow rate: 0.8 mL / min, injection volume: 5 μL, mobile phase buffer: 20 mM KH2PO4 (adjusted to pH 2.0 with phosphoric acid).

[0032] Example 2 The nitrile hydrolase RrNI sequence is shown in SEQ ID NO:4, source: Rhodococcus rhodochrous J3. Nitrile hydrolase G The sNI sequence is shown in SEQ ID NO:5, originating from Gordonia sputi NBRC 100414. Nitrile hydrolase G The nNI sequence is shown in SEQ ID NO:6, originating from Gordonia namibiensis NBRC 108229. The three target amino acid sequences obtained from the screening (RrNI, ... G sNI、 GThe gene sequence (nNI) was submitted to Changzhou Xinyisheng Life Technology Co., Ltd., who were commissioned to optimize the codons according to the codon preferences of *E. coli*. The gene was synthesized using high-throughput technology and cloned into the NdeI / XhoI sites of the pET28a+ vector using double enzyme digestion and ligation techniques for plasmid construction. After cloning the optimized gene sequence into the pET28a+ plasmid, sequencing was performed to verify sequence accuracy. The constructed plasmid will be used for subsequent heterologous expression experiments.

[0033] Nitrile hydrolase plasmid transformation, protein expression, and purification: Plasmid transformation: 2 μL of the constructed plasmid was added to 100 μL of E. coli BL21(DE3) competent cells on a clean bench. After incubation on ice for 30 min, the cells were heat-shocked in a 42°C water bath for 45 s, followed by incubation on ice for 2 min. 700 μL of LB liquid medium was added, and the cells were incubated on a shaker at 37°C and 200 rpm for 1 h. The mixture was then spread onto LB solid medium containing 50 μg / mL kanamycin sulfate and cultured at 37°C for 12 h.

[0034] Protein expression: Single colonies were selected and cultured in 5 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate at 37°C on a shaker at 200 rpm until turbidity was observed. The culture was then transferred to a large-volume medium at a 1% inoculum size and cultured under the same conditions. When the OD600 reached 1.1–1.5, 0.5 mM IPTG was added, and the cells were induced at 25°C and 180 rpm for 16 h. After centrifugation at 12000 rpm and 4°C for 20 min, the supernatant was discarded, and the cells were washed with 50 mM Tirs-H2SO4 (pH=8.2) buffer and centrifuged again to obtain whole bacterial cells, which were then stored at -80°C.

[0035] Purification: The collected whole bacterial cells were added to 0.1M phosphate buffer (pH 7.6) containing 25 mM imidazole. After sonication in an ice-water bath for one hour, the cells were centrifuged at 12,000 rpm for 20 min at 4°C. The supernatant was collected using a syringe and filtered through a 0.22 μm filter. The supernatant was stored on ice. The preservation solution in the nickel column was replaced with ultrapure water. The nickel column was equilibrated with 0.1M phosphate buffer (pH 7.6) containing 25 mM imidazole. The sample was slowly loaded onto the column using a dropper and incubated on ice for 0.5 h to allow the protein to fully bind to the nickel column. Impurities were eluted with 50 mM imidazole phosphate buffer (pH 7.6), and the target protein was eluted with 250 mM imidazole 0.1M phosphate buffer (pH 7.6). The eluent was collected in separate tubes and labeled. The protein eluent was transferred to ultrafiltration tubes and ultrafiltered at 3900 rpm using a horizontal centrifuge at 4°C to desalt and concentrate the protein sample. After ultrafiltration, the protein sample was added to a final concentration of 20% glycerol, and the protein concentration was determined using the Coomassie Brilliant Blue method. The sample was then flash-frozen in liquid nitrogen and stored at -80°C.

[0036] Three nitrile hydrolases were expressed and purified using the methods described above, and then analyzed by SDS-PAGE electrophoresis. like Figure 4 As shown, electrophoretic analysis revealed that GsNI exhibited an extremely dense and clear single target main band; simultaneously, RrNI and GnNI also showed obvious target protein bands, with apparent molecular weights basically consistent with theoretical expectations. The target protein bands in each lane were clearly characterized, proving that this embodiment successfully achieved efficient expression and purification of three nitrile hydrolases, and the purity and concentration of the obtained proteins met the requirements of subsequent experiments.

[0037]

[0038] The total reaction volume was 1 mL, and the system consisted of the following: 3-cyanopropionic acid (50 mM), purified enzyme solution (any one of nitrile hydrolase RrNI, nitrile hydrolase GsNI, or nitrile hydrolase GnNI, 100 nM), and PB buffer (pH = 5.6). The concentrations represent the final feed concentrations of each reactant. The reaction mixture was carried out on a shaker (30°C, 700 rpm) for 24 hours.

[0039] Control experiment: The reaction system is the same as above, but no nitrile hydrolase is added to the control group.

[0040] The yield of succinic acid was analyzed by high-performance liquid chromatography (HPLC). The detection conditions were as follows: Horizon AQUA C18 5u HPLC column (catalog number: AQUA-250-46-50), temperature: 25℃, flow rate: 1 mL / min, injection volume: 5 μL, mobile phase buffer: a mixture of methanol and potassium dihydrogen phosphate solution (0.01 mol / L, pH 2.1) at a volume ratio of 5:95. The reaction results are shown in Table 2. Table 2

Claims

1. A method for in vitro biosynthesis of succinic acid, comprising the steps of: 3-Cyanopyranoic acid is mixed with the first enzyme in the first buffer solution to obtain the first mixture, and the reaction yields succinic acid. 。 2. The method for in vitro biosynthesis of succinic acid according to claim 1, characterized in that, The first enzyme is selected from proteins that have more than 90% homology with the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 and have nitrile hydrolase function.

3. The method for in vitro biosynthesis of succinic acid according to claim 1, characterized in that, Each 1L of the first buffer solution contains 40-60 mmol of 3-cyanopropionic acid.

4. The method for in vitro biosynthesis of succinic acid according to claim 1, characterized in that, Each 1L of the first buffer solution contains 0.080~0.12mmol of the first enzyme.

5. The method for in vitro biosynthesis of succinic acid according to claim 1, characterized in that, The first buffer solution is PB buffer solution; and / or, the reaction temperature is 25~37℃.

6. The method according to claim 1, characterized in that, The method for synthesizing the substrate 3-cyanopropionic acid is as follows: L-glutamic acid, H2O2, haloperoxidase VHPOs, second buffer solution, and NaBr were mixed to obtain a second mixture, and the reaction yielded 3-cyanopropionic acid. 。 7. The method for in vitro biosynthesis of succinic acid according to claim 6, characterized in that, The halogen peroxidases VHPOs are selected from proteins that have more than 90% homology with the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO:

3.

8. The method for in vitro biosynthesis of succinic acid according to claim 6, characterized in that, Each 1L of the second mixture contains 0.08~0.12mmol of L-glutamic acid.

9. The method for in vitro biosynthesis of succinic acid according to claim 6, characterized in that, Each 1L of the second mixture contains 0.4~0.6mol of H2O2.

10. The method for in vitro biosynthesis of succinic acid according to claim 6, characterized in that, The second buffer is a sodium citrate buffer with a pH of 5 to 7; and / or, each 1 L of the second buffer contains 0.4 to 0.6 mmol of NaBr.

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