Method for preparing medium oxalic acid through biological catalysis
Through biocatalytic methods, 1,3-dihydroxyacetone was converted into mesooxalic acid using recombinant E. coli and succinate semialdehyde dehydrogenase, which solved the pollution problem of precious metal catalysts and organic solvents in mesooxalic acid production, and achieved efficient, safe and environmentally friendly mesooxalic acid preparation.
Patent Information
- Application Number
- CN202311417353.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-10-30
- Publication Date
- 2025-05-02
AI Technical Summary
The production process of oxalic acid usually requires the use of precious metal catalysts and organic solvents, which leads to environmental pollution, low process safety and high cost, limiting its application.
Using biocatalytic methods, 1,3-dihydroxyacetone was converted into methoxalic acid under mild conditions by using recombinant E. coli and succinate semialdehyde dehydrogenase expressing galactose oxidase.
It has achieved efficient preparation of oxalic acid, which is simple, safe and non-toxic, does not produce harmful by-products, and has good industrial application prospects.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of biocatalysis, and in particular relates to a method for preparing oxalic acid through biocatalysis. Background Art
[0002] Mesoxalic acid (2-Ketomalonic acid), also known as 2-ketomalonic acid, has a CAS number of 473-90-5 and a molecular weight of 118.04. Mesoxalic acid, as a highly functional molecule, has potential uses as a complexing agent and precursor in organic synthesis, and its chlorophenylhydrazone has relevant biological activity as an anti-HIV drug.
[0003] In 1997, Fordham et al. reported that glycerol was first oxidized to synthesize glyceric acid at 80°C, pH 5.0 and the catalyst PtBi / C, then tartaric acid was obtained, and then further oxidized to obtain mesoxalic acid (Fordham P, Besson M, Gallezot P. Catalytic oxidation with air of tartronic acid to mesoxalic acid on bismuth-promoted platinum. Catalysis Letters, 1997, 46(3): 195-199). In 2003, Ciriminna et al. reported the selective conversion of glycerol to ketomalonic acid at pH 10.0 using NaOCl as a regenerative oxidant in water at 2°C in the presence of a catalyst of sodium bromide and TEMPO (2,2,6,6-tetramethylpiperidinyl oxide) (Ciriminna Rosaria, Pagliaro M. One-Pot Homogeneous and heterogeneous oxidation of glycerol to ketomalonic acid mediated by TEMPO. Advanced Synthesis & Catalysis, 2003, 345: (3): 383-388). In 2019, Hong et al. reported that mesoxalic acid can be prepared by selective oxidation of glycerol using the laccase / TEMPO system. The first step in the reaction is that glycerol first generates glyceraldehyde, the second step is that glyceraldehyde is converted into glyceric acid, the third step is that glyceric acid is converted into tartaric acid, and the fourth step is the oxidative dehydrogenation of tartaric acid to generate mesoxalic acid (Hong CS, Chin SY, Cheng CK, Chua GK. Selective oxidation of glycerol to mesoxalic acid by laccase. Chemical Engineering Communications, 2019, 206(12): 1645-1660).
[0004] According to the current reports on the production and preparation of meso-oxalic acid, the production of meso-oxalic acid mainly adopts chemical methods, and the production process usually requires the use of precious metal catalysts, organic solvents, etc., which has problems such as environmental pollution and low process safety, which also leads to the current high cost of meso-oxalic acid and limits its application. Therefore, it is necessary to propose a new synthesis method, which has important economic significance for further improving the production technology of the product. Summary of the invention
[0005] The invention discloses a method for preparing oxalic acid by biocatalysis, which solves the problems that noble metal catalysts and organic solvents are usually needed in the current oxalic acid production process, and there are environmental pollution and low process safety.
[0006] The technical solution of the present invention is as follows:
[0007] A method for preparing oxalic acid by biocatalysis, the method comprising adding recombinant Escherichia coli cells expressing galactose oxidase to a buffer solution containing 1,3-dihydroxyacetone, and then performing an oscillation reaction. After the oscillation reaction is completed, a reaction solution is obtained, and then the reaction solution is centrifuged, the supernatant is taken, the pH is adjusted with sodium hydroxide, and then succinic semialdehyde dehydrogenase and NAD are added. + , and react by shaking at 30° C. to obtain a reaction solution containing meso-oxalic acid, and the meso-oxalic acid is extracted using the reaction solution containing meso-oxalic acid.
[0008] Furthermore, the gene sequence of the galactose oxidase is shown in SEQ ID No.1, and the gene sequence of the succinate semialdehyde dehydrogenase is shown in SEQ ID No.2.
[0009] Furthermore, the method for obtaining the recombinant Escherichia coli cells expressing galactose oxidase is:
[0010] Step 11: constructing a genetically engineered strain expressing galactose oxidase;
[0011] Step 12: Express galactose oxidase.
[0012] Furthermore, the specific method for constructing a genetically engineered strain expressing galactose oxidase is to construct the galactose oxidase gene sequence into the plasmid pET-28a(+) to obtain the recombinant plasmid pET28a-GO; and transform the constructed recombinant plasmid pET28a-GO into Escherichia coli BL21(DE3) to obtain an engineered Escherichia coli strain capable of expressing galactose oxidase.
[0013] Furthermore, the specific method for expressing galactose oxidase is to inoculate the engineered Escherichia coli strain expressing galactose oxidase constructed in step 11 into a shake flask containing LB liquid culture medium, and after inoculation, culture it in a shaker at 37°C until the OD600 of the bacteria is 0.6, add IPTG for induction, continue to culture it in a shaker at 16°C, and then collect the engineered Escherichia coli cells expressing galactose oxidase in the culture medium by centrifugation, wash the cells twice, resuspend the washed cells in phosphate buffer to obtain a bacterial solution, add CuSO4 to the resuspended bacterial solution, and obtain recombinant Escherichia coli cells expressing galactose oxidase.
[0014] Furthermore, the method for obtaining the succinate semialdehyde dehydrogenase is:
[0015] Step 21: constructing a genetically engineered strain expressing succinate semialdehyde dehydrogenase;
[0016] Step 22: Express and purify succinate semialdehyde dehydrogenase.
[0017] Furthermore, the specific method for constructing a genetically engineered strain expressing succinate semialdehyde dehydrogenase is to construct the succinate semialdehyde dehydrogenase gene sequence into plasmid pETDuet-1 to obtain a recombinant plasmid pETDuet-sad; and transform the constructed recombinant plasmid pETDuet-sad into Escherichia coli BL21 (DE3) to obtain an engineered Escherichia coli strain expressing succinate semialdehyde dehydrogenase.
[0018] Furthermore, the specific method for expressing and purifying succinic semialdehyde dehydrogenase is as follows: the engineered Escherichia coli strain expressing succinic semialdehyde dehydrogenase constructed in step 21 is inoculated into a shake flask containing LB liquid culture medium, and after inoculation, the strain is cultured in a shaking incubator at 37°C until the OD600 of the strain is 0.6, IPTG is added for induction, and the shaking incubator is continued at 16°C, and the engineered Escherichia coli strain expressing succinic semialdehyde dehydrogenase in the culture medium is collected by centrifugation, the strain is washed twice with sodium phosphate buffer to collect the strain, and then the strain is crushed under high pressure, and the succinic semialdehyde dehydrogenase is purified using a nickel column and stored in a sodium phosphate buffer.
[0019] Furthermore, the buffer solution is a phosphate buffer solution, and the pH of the phosphate buffer solution is 7.4; the pH is adjusted to 8.0 using sodium hydroxide.
[0020] The present invention provides a method for preparing oxalic acid by biocatalysis, wherein 1,3-dihydroxyacetone is used as a raw material, and galactose oxidase-expressing engineered Escherichia coli and aldehyde dehydrogenase are used for biocatalysis to prepare oxalic acid. The method for preparing oxalic acid by the present invention has a simple process, mild and non-toxic synthesis reaction conditions, no harmful byproducts, high process safety, and good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 : Pathway for biocatalytic synthesis of oxalic acid using 1,3-dihydroxyacetone as raw material;
[0022] Figure 2 : Electrophoresis of purified succinate semialdehyde dehydrogenase;
[0023] Figure 3: Liquid quality detection diagram of oxalic acid; the numbers marked from left to right in the reaction solution-MS diagram are 96.9475, 116.9573, 179.8987, 194.9055, 216.8833, 292.8644, 314.8423, 336.8200, 390.8237, 488,7927; the numbers marked from left to right in the standard sample-MS diagram of oxalic acid are 116.9659 and 134.9740;
[0024] Figure 4 : Liquid chromatogram of oxalic acid after derivatization with o-phenylenediamine. DETAILED DESCRIPTION
[0025] Terminology explanation:
[0026] Deoxyribonucleic acid: DNA
[0027] Galactose oxidase: GO
[0028] Succinate semialdehyde dehydrogenase: Sad
[0029] Nicotinamide Adenine Dinucleotide: NAD +
[0030] Escherichia coli: E.coli
[0031] "Expression" refers to the expression of a specific gene in an organism in large amounts.
[0032] The present invention is further illustrated below by specific examples, but the present invention is not limited to the following embodiments.
[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0034] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
[0035] The enzyme reagents used were purchased from Thermo Fisher Scientific (China) Co., Ltd., the kits used to extract plasmids and recover DNA fragments were purchased from OMEGA, USA, and the cloning kits were purchased from Beijing Quanshijin Biotechnology Co., Ltd. The corresponding operation steps were carried out according to the product instructions; all culture media were prepared with deionized water unless otherwise specified.
[0036] Culture medium formula:
[0037] 1) Solid LB medium
[0038] Solid LB medium: 5g / L yeast powder, 10g / L tryptone, 10g / L NaCl, 15g / L agar powder, the rest is water, sterilize at 121℃ for 20min, add ampicillin when using.
[0039] 2) Liquid LB medium
[0040] LB medium: 5 g / L yeast powder, 10 g / L tryptone, 10 g / L NaCl, the rest is water, sterilized at 121°C for 20 min.
[0041] During the culture process, a certain concentration of antibiotics may be added to the culture medium to maintain the stability of the plasmid, such as 50 μg / mL of kanamycin.
[0042] 3) Sodium phosphate buffer
[0043] Prepare 1M solution of disodium hydrogen phosphate and sodium dihydrogen phosphate, then take 93.2mL of disodium hydrogen phosphate solution and 6.8mL of sodium dihydrogen phosphate solution, add water to 1L, and adjust the pH to 8.0 to obtain 100mM sodium phosphate buffer.
[0044] 4) Phosphate buffer (1L)
[0045] NaCl, 8g; KCl, 0.2g; Na2HPO4, 1.44g; KH2PO4, 0.24g, adjust the pH to 7.4 with hydrochloric acid.
[0046] 5) Acetate buffer
[0047] Prepare 200 mM acetic acid solution (A) and 200 mM sodium acetate solution (B), then take 82 ml of acetic acid solution and 18 ml of sodium acetate solution, stir and mix well to obtain 200 mM acetate buffer.
[0048] Example 1 Construction and expression of genetically engineered strains of galactose oxidase
[0049] Using oligonucleotides 5'-CGCGGATCCATGGCCAGTGCGCCGATCGG-3' (SEQ ID No. 3) and 5'-GCGTCGACTTACTGGGTAACACGAATCG-3' (SEQ ID No. 4) as primers and chemically synthesized galactose oxidase gene as template, the GO gene was amplified by polymerase chain reaction (PCR) method, and BamHI and SalI sites were introduced at the 5' and 3' ends respectively, and then the gene was cloned into the pET-28a (+) vector using the above restriction sites to obtain the recombinant plasmid pET28a-GO. The constructed recombinant plasmid pET28a-GO was transformed into Escherichia coli BL21 (DE3) competent cells, and coated on solid LB medium plates to obtain an engineered Escherichia coli strain capable of expressing galactose oxidase.
[0050] Select a single colony on the plate and inoculate it into 20 mL of LB liquid medium containing the corresponding antibiotics. Cultivate it for about 12 hours as a seed solution. Inoculate it into LB liquid medium containing the corresponding antibiotics at a 1% inoculum volume. After inoculation, incubate it in a shaking incubator at 37°C until the bacterial OD reaches 0. 600 The pH value was about 0.6, IPTG was added for induction, and the culture was continued at 16°C in a shaking incubator. The engineered E. coli cells in the culture medium were collected by centrifugation at 8000 rpm at 4°C for 10 min, and the cells were washed twice. The culture medium was resuspended with 10 mM phosphate buffer (pH 7.4), and CuSO4 was added with a final concentration of 0.5 mM, and activated at 4°C for 48 h.
[0051] Example 2 Construction of a recombinant plasmid expressing succinate semialdehyde dehydrogenase
[0052] (1) Construction of pETDuet-sad plasmid
[0053] The PCR primer sequences used in this example are listed in Table 2. The succinic semialdehyde dehydrogenase gene sad (gene sequence as shown in SEQ ID NO. 2, derived from the Escherichia coli genome, GenBank: 947440) was amplified using primers sad-F and sad-R; PCR amplification was performed using primers pETDut-F and pETDut-R, and plasmid pETDuet-1 was linearized at the MCS site. After purification with a DNA purification kit, the gene sad and the linearized pETDuet-1 fragment were cloned in one step using Basic Assembly Mix (Beijing Quanshijin Biotechnology Co., Ltd., catalog number: CU201-02) (the one-step cloning system is shown in Table 1). The one-step cloning system directly heat-shocked the competent cells of Escherichia coli BL21 (DE3), and the transformation product was evenly spread on an LB plate containing 100 μg / mL ampicillin. After culturing at 37°C overnight, transformants containing the recombinant expression plasmid pETDuet-sad were obtained; single clones were picked for sequencing verification (using primer pairs UP1 and T7) to obtain recombinant genetically engineered bacteria.
[0054] PCR reaction system (50 μL reaction system): 1 μL E. coli solution (or plasmid pETDuet-1) as template, 1 μL of primer F and primer R (Table 2) at a concentration of 10 μM, Max Master Mix high-fidelity DNA polymerase 25μL, ultrapure water 22μL.
[0055] PCR reaction conditions: pre-denaturation at 98°C for 5 min, then enter temperature cycle at 98°C for 15 s; 60°C for 15 s; 72°C for 45 s (or 90 s); a total of 30 cycles, 72°C, final extension for 5 min, and the final termination temperature is 12°C.
[0056] Table 1 One-step cloning system to construct pETDuet-sad
[0057]
[0058] Table 2 Construction of recombinant plasmids and related primers for verification
[0059]
[0060] Example 3 Inducible expression and protein purification of succinate semialdehyde dehydrogenase
[0061] The engineered bacteria obtained in Example 2 were inoculated into 10 mL of liquid LB, ampicillin was added, and cultured at 37°C, 200 rpm overnight. 1% of the bacteria were inoculated into 200 mL of LB and cultured at 37°C, 200 rpm until the OD 600The concentration of IPTG was 0.6-0.8, and a final concentration of 0.5 mM was added for induction. The culture was carried out at 16°C and 200 rpm for about 20 hours, and the bacterial solution was placed in a centrifuge cup and centrifuged at 4°C and 8000 rpm for 10 minutes. The supernatant was discarded, and the bacterial cells were washed twice with sodium phosphate buffer. Finally, the bacterial cells were collected and concentrated with pure water to OD 600 The cell was disrupted by a high pressure cell disruptor, and then the protein was purified by a nickel column and stored in sodium phosphate buffer. The electrophoresis of purified succinic semialdehyde dehydrogenase was as follows: Figure 2 shown.
[0062] Example 4 Biocatalytic Preparation of Oxalic Acid
[0063] The activated bacterial solution obtained in Example 1 was diluted with 10 mM phosphate buffer (pH 7.4) to a bacterial OD of 600 The reaction temperature was 20, the concentration of 1,3-dihydroxyacetone was 0.3 M, the total volume was 1 L, and the reaction was carried out at 200 rpm at 20-40 °C for 24 h.
[0064] The supernatant was then centrifuged and the pH was adjusted to 8.0. Purified succinic semialdehyde dehydrogenase was added and reacted at 30°C for 2 h. The specific reaction system was: 250 μL supernatant, succinic semialdehyde dehydrogenase, NAD + (80 mM) 250 μL, add sodium phosphate buffer to 1 mL.
[0065] Subsequently, 50 mL of the reaction solution was taken into a 250 mL flask, and sodium hydroxide solution was used to adjust the alkalinity and freeze-dried to obtain a white solid. The white solid was taken, ethanol and p-toluenesulfonic acid were added for esterification, and the reaction was carried out at 100 ° C for 15 hours. The organic phase was extracted as diethyl ketomalonate after washing with water. The obtained diethyl ketomalonate was then added to a sodium hydroxide ethanol solution for reaction. After TLC (iodine fumigation) showed that there was no residual raw material, the reaction solution was filtered and dried to obtain a white solid. The solid was dissolved with hydrochloric acid, reacted at room temperature, concentrated, and fully dissolved with methyl tert-butyl ether and filtered. The filtrate was dried and concentrated to obtain meso-oxalic acid.
[0066] Detection of oxalic acid in Example 5
[0067] After the reaction is completed, centrifuge 1 mL of the reaction solution, take 300 μL of the supernatant, add 100 μL of 6 g / L o-phenylenediamine, react at 35°C in the dark for 30 min, and add 100 μL of 0.2 M acetate buffer. Centrifuge, take the supernatant, filter through a 0.22 μm filter membrane, and use a liquid chromatograph and a liquid chromatography-mass spectrometer to detect oxalic acid. The liquid chromatography-mass spectrometry detection diagram of oxalic acid is shown in the figure. Figure 3 As shown in the figure, the liquid chromatogram of oxalic acid after being derivatized with o-phenylenediamine is as follows Figure 4The molecular weight of oxalic acid is 117.99, and an ion peak of 116.99 is generated in the negative ion mode of mass spectrometry, as shown in Figure 3 As shown, the reaction solution sample contains an ion peak with a molecular weight of 116.95. The oxalic acid standard is detected using the same method and also produces an ion peak with a molecular weight of 116.95. Therefore, it is inferred that the reaction solution contains oxalic acid in the product; oxalic acid is detected by o-phenylenediamine derivatization, and 3-hydroxy 2-carboxyquinoxaline is produced after derivatization. The liquid chromatography results are shown as follows Figure 4 As shown, the retention time is 7.5 min.
[0068] Liquid chromatography conditions: detector: UV detector; chromatographic column: Agilent ZORBAX SB-C18 (4.6×250mm); detection wavelength: 272nm; column temperature: 30℃; flow rate: 0.5mL / min; injection volume: 10μL; mobile phase: A is 0.1% acetic acid aqueous solution, mobile phase B is methanol; gradient elution program: 0-5min, 30% B solution; 5-10min, linear change to 90% B solution; 10-15min, 90% B solution; 15-20min, linear change to 30% B solution; 20-25min, 30% B solution.
[0069] LC-MS conditions: chromatographic column: Eclipse Plus C18 column (1.8μm, 50mm×2.1mm); column temperature: 35℃; flow rate: 0.2mL / min; injection volume: 2μL; mobile phase: A is water, mobile phase B is acetonitrile; gradient elution program: 0-5min, linear change from 10% B liquid to 60% B liquid; 5-7min, linear change to 100% B liquid; 7-8min, 100% B liquid; 8-9min, 10% B liquid. Gas temperature: 300℃; gas flow rate: 7L / min; nebulizer: 35psi; protective gas temperature: 325℃; protective gas flow rate: 11L / min; capillary: 3000V.
[0070] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.
Claims
1. A method for preparing oxalic acid by biocatalysis, characterized in that: The method comprises adding recombinant Escherichia coli cells expressing galactose oxidase to a buffer solution containing 1,3-dihydroxyacetone, and then performing a shaking reaction. After the shaking reaction is completed, a reaction solution is obtained, and then the reaction solution is centrifuged, the supernatant is taken, the pH is adjusted with sodium hydroxide, and then succinic semialdehyde dehydrogenase and NAD are added. + , and react by shaking at 30° C. to obtain a reaction solution containing meso-oxalic acid, and the meso-oxalic acid is extracted using the reaction solution containing meso-oxalic acid.
2. The method according to claim 1, characterized in that The gene sequence of the galactose oxidase is shown as SEQ ID No.1, and the gene sequence of the succinate semialdehyde dehydrogenase is shown as SEQ ID No.
2.
3. The method according to claim 1, characterized in that The method for obtaining the recombinant Escherichia coli cells expressing galactose oxidase is: Step 11: constructing a genetically engineered strain expressing galactose oxidase; Step 12: Express galactose oxidase.
4. The method according to claim 3, characterized in that The specific method for constructing a genetically engineered strain expressing galactose oxidase is to construct a galactose oxidase gene sequence onto a plasmid pET-28a(+) to obtain a recombinant plasmid pET28a-GO; and transform the constructed recombinant plasmid pET28a-GO into Escherichia coli BL21(DE3) to obtain an engineered Escherichia coli strain capable of expressing galactose oxidase.
5. The method according to claim 3, characterized in that: The specific method for expressing galactose oxidase is as follows: the engineered Escherichia coli strain expressing galactose oxidase constructed in step 11 is inoculated into a shaking flask containing LB liquid culture medium, and after inoculation, it is cultured in a shaking incubator at 37°C until the OD600 of the bacteria reaches 0.5-1.0, IPTG is added for induction, and the shaking incubator is continued at 16°C, and then the engineered Escherichia coli cells expressing galactose oxidase in the culture medium are collected by centrifugation, the bacteria are washed twice, and the washed bacteria are resuspended in phosphate buffer to obtain a bacterial solution, and CuSO4 is added to the resuspended bacterial solution to obtain recombinant Escherichia coli cells expressing galactose oxidase.
6. The method according to claim 1, characterized in that The method for obtaining the succinate semialdehyde dehydrogenase is: Step 21: constructing a genetically engineered strain expressing succinate semialdehyde dehydrogenase; Step 22: Express and purify succinate semialdehyde dehydrogenase.
7. The method according to claim 6, characterized in that The specific method for constructing a genetically engineered strain expressing succinate semialdehyde dehydrogenase is as follows: constructing a succinate semialdehyde dehydrogenase gene sequence onto a plasmid pETDuet-1 to obtain a recombinant plasmid pETDuet-sad; and transforming the constructed recombinant plasmid pETDuet-sad into Escherichia coli BL21 (DE3) to obtain an engineered Escherichia coli strain expressing succinate semialdehyde dehydrogenase.
8. The method according to claim 6, characterized in that The specific method for expressing and purifying succinic semialdehyde dehydrogenase is as follows: the engineered Escherichia coli strain expressing succinic semialdehyde dehydrogenase constructed in step 21 is inoculated into a shaking flask containing LB liquid culture medium, and then cultured in a shaking incubator at 37° C. until the bacterial OD reaches 0. 600 When the pH reaches 0.5-1.0, IPTG is added for induction, and the shaking culture is continued at 16°C. The engineered Escherichia coli cells expressing succinic semialdehyde dehydrogenase in the culture medium are collected by centrifugation, and the cells are washed twice with sodium phosphate buffer to collect the cells, and then the cells are crushed under high pressure, and the succinic semialdehyde dehydrogenase is purified using a nickel column and stored in sodium phosphate buffer.
9. The method according to claim 1, characterized in that: The buffer is a phosphate buffer, and the pH of the phosphate buffer is 7.4; the pH is adjusted to 8.0 with sodium hydroxide.