A kind of enzymatic synthesis process of isocitrate

Abstract

The invention discloses an enzymatic synthesis process of isocitric acid, belonging to the field of drug intermediate enzyme catalysis. Comprise the following steps: step one, the conversion of isocitric acid, in the reaction system, citric acid, water and aconitase solution are respectively added to form a reaction mixture, water bath isothermal reaction, and isocitric acid reaction solution is obtained; during the reaction process, aconitase catalyzes citric acid into isocitric acid; step two, the purification of isocitric acid, the isocitric acid reaction solution is heated and activated carbon is added to adsorb impurities, and the clear liquid is filtered to obtain the clear liquid, and the clear liquid continuously rinses the ion exchange resin column, and the ion exchange resin column is washed after the rinsing is completed, and the ion exchange resin column is de-washed after the washing is completed, and the eluent is cooled and crystallized, and the potassium isocitrate finished product is obtained after solid-liquid separation and drying. The present invention is a kind of synthesis process that is convenient for preparing isocitric acid and prepares a high purity product.

Description

Technical Field

[0001] The invention mainly relates to the technical field of organic chemical raw materials, and specifically to an enzymatic synthesis process of isocitric acid. Background Art

[0002] Isocitrate is an isomer of citric acid. Although it is present in small quantities, it is widely present in the biological world. As a raw material, isocitrate has broad application prospects in the fields of medicine and chemical industry.

[0003] The synthesis of tetrahydrofuranol uses potassium isocitrate as raw material, which is crucial in the synthesis of a variety of clinical HIV protease inhibitors (such as darunavir, brenavir, GS-9005 and SPI-256), and has the effect of preventing and treating HIV / AIDS.

[0004] According to the citric acid preparation method provided by the patent document with application number CN201410590425.2, the raw material used in the preparation method is orange, which is obtained by juicing, salt precipitation, alcohol dissolution, distillation and drying; salt precipitation is to heat the orange juice to 75-85°C, add an excess of calcium chloride saturated solution, filter, precipitate as calcium citrate, and wash with hot water 3-5 times; alcohol dissolution is to put calcium citrate into a reactor with a stirring device, add concentrated sulfuric acid with a mass of 0.3-0.6 times of calcium citrate, stir into a paste, then add ethanol, and stir and react for 3-5 hours; the distillation temperature is 80-85°C; the drying temperature is 60-70°C, and the drying time is 1-3 hours. The preparation method has a short process flow, low equipment investment, and is easy to operate.

[0005] The preparation method in the above patent has a short process flow, low equipment investment, and is easy to operate, but is unable to prepare isocitric acid, and the purity of the prepared product is low. Summary of the invention

[0006] The present invention mainly provides an enzymatic synthesis process of isocitrate, which is used to solve the technical problems raised in the above background technology.

[0007] The technical solution adopted by the present invention to solve the above technical problems is:

[0008] An enzymatic synthesis process of isocitrate comprises the following steps:

[0009] Step 1: Conversion of isocitric acid: in the reaction system, citric acid, water and aconitase solution are added to form a reaction mixture, and the mixture is reacted in a water bath at a constant temperature to obtain an isocitric acid reaction solution.

[0010] Step 2: Purification of isocitric acid. The isocitric acid reaction solution is heated and activated carbon is added to adsorb impurities. The clear liquid is filtered to obtain a clear liquid. The clear liquid is used to continuously rinse the ion exchange resin column. After the rinsing is completed, the ion exchange resin column is washed with water. After the washing is completed, the ion exchange resin column is de-eluted. The eluate is cooled and crystallized, and the potassium isocitrate product is obtained after solid-liquid separation and drying.

[0011] Preferably, the method for obtaining the aconitase solution in step 1 is: using Escherichia coli BL21 (DE3) as a host strain, heterologously expressing the Cit gene encoding aconitase from Bacillus, and obtaining an Escherichia coli engineered strain capable of catalyzing citric acid into isocitric acid; then obtaining the aconitase solution through fermentation culture and cell disruption treatment; the nucleotide sequence of the expression vector of the engineered strain contains the gene sequence described in SEQ ID NO.1.

[0012] Preferably, the method for obtaining the aconitase solution in step 1 is: designing a pair of primers according to the Bacillus aconitase gene sequence disclosed on GenBank; using Bacillus genomic DNA as a template, PCR amplifying the Cit gene fragment, double-digesting and purifying the PCR-amplified gene fragment and the pET-28 vector, establishing an expression vector pET-28-Cit connected to the Cit encoding gene, adding Escherichia coli BL21 (DE3) competent cells to transform and construct an engineered strain, spreading the engineered strain on a kanamycin-resistant plate for culture, and obtaining the aconitase solution through fermentation culture and cell disruption.

[0013] Preferably, the initial reaction concentration of citric acid in step 1 is 50-150 g / L, preferably 80-120 g / L.

[0014] Preferably, during the reaction in step 1, the pH value is adjusted to be constant at 7.0-7.6 (preferably 7.2-7.4) using 0.5-2.5 mol / L (preferably 1-1.2 mol / L) potassium hydroxide solution.

[0015] Preferably, the amount of aconitase added in step 1 is 1-5%, preferably 2-3% of the reaction system.

[0016] Preferably, the catalytic reaction time in step 1 is 8-14 h, preferably 10-12 h; the reaction temperature is 30-45°C, preferably 35-40°C.

[0017] Preferably, in step 2, the heating temperature is 55-85°C, preferably 65-75°C; the amount of activated carbon added is 0.5-3%, preferably 1-2%; and the insulation time is 0.5-1.5h, preferably 1h.

[0018] Preferably, when the ion exchange resin column is de-eluted in step 2, the de-eluting agent is potassium hydroxide at a concentration of 10-30% (preferably 15-25%).

[0019] Preferably, in step 2, the crystallization condition is 0-5°C (preferably 3-4°C), and the crystallization time is 12-18h (preferably 14-16h).

[0020] Preferably, the drying temperature in step 2 is 50-60° C. (preferably 55° C.) and the drying time is 4-8 h (preferably 5-7 h).

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) This method provides a new method for preparing isocitrate, namely, a bio-enzyme catalysis method, which uses citric acid as a substrate and utilizes aconitase to biosynthesize isocitrate.

[0023] (2) The enzymatic catalysis using the aconitase of the present invention has a high yield and a high purity of the prepared product, and the conversion rate can reach more than 80%; it plays an important role as an intermediate in the synthesis of various clinical HIV protease inhibitors (such as darunavir, brenavir, GS-9005 and SPI-256);

[0024] (3) The method of the present invention is simple to operate, low in cost, and has little discharge of three wastes, etc. It is suitable for industrial production and has good market competitiveness.

[0025] The present invention will be explained in detail below with reference to the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The expression plasmid pET-28a-Cit constructed by the present invention;

[0027] Figure 2 This is the HPLC spectrum of potassium isocitrate prepared in Example 2;

[0028] Figure 3 The present invention is an experimental device for preparing potassium isocitrate;

[0029] Figure 4 The present invention is an experimental device for purifying potassium isocitrate;

[0030] Figure 5 It is a schematic diagram of the synthesis reaction formula of the present invention;

[0031] In the figure: 10, synthesis box; 22, telescopic cylinder; 27, vibration plate; 25, vibration motor; 24, vibration plate; 26, connecting rod; 28, storage box; 32, drive motor; 34, stirring turntable; 33, cylinder; 13, filter port; 43, connecting pipe; 41, elution box; 45, clamping plate; 411, sewage pipe; 42, partition; 46, de-washing agent tank; 412, discharge pipe. DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0033] Example 1: Preparation of aconitase solution

[0034] 1. Construction of aconitase cloning strain

[0035] After the Bacillus at -80°C was revived and subcultured, the genome was extracted as a DNA template, and a pair of primers were designed according to the sequence of the Bacillus aconitase gene (such as GenBank: AQR86003.1) published on NCBI: forward primer (Cit-F-BamHI): 5'-CGCGGATCCATGGCAAACGAGCAAAAA-3'; reverse primer (Cit-R-Xho I): 5'-CCGCTCGAGCAGGACTGCTTCATTTTTTC-3'; PCR amplification, gel verification and product recovery after PCR. Electrophoresis detection and gel cutting to recover the target fragment, the recovered target protein was double-digested with BamHI-Xho I, and connected with the pET-28a plasmid that was also double-digested with BamHI-Xho I at 16°C overnight, and the expression vector pET-28a-Cit encoding the Cit gene was obtained after connection.

[0036] 2. Construction of aconitase expression strain

[0037] The expression vector pET-28a-Cit encoding the Cit gene was heat-shock transformed into Escherichia coli BL21 (DE3) competent cells, and positive transformants were screened on kanamycin resistance plates, PCR and double enzyme digestion verification were performed, and the verified correct transformants were fermented and cultured, and cell disruption was performed to obtain aconitase enzyme solution.

[0038] 3. Preparation of aconitase solution

[0039] The E. coli engineered bacteria prepared in step 1 were inoculated onto a slant containing kanamycin resistance, and after culturing at 37°C for 16 hours, a loop was picked from the slant with an inoculation loop and transferred to a TB medium containing kanamycin resistance, and cultured at 37°C and 200rpm on a shaking table until OD600=0.6, and inoculated into a fermentation medium containing kanamycin resistance with an inoculum of 1% by volume concentration, and cultured at 37°C and 200rpm on a shaking table until OD600=0.5, and then IPTG with a final concentration of 3mM was added and cultured at 24°C and 200rpm for 16 hours, and the fermentation broth was centrifuged at 4°C and 5000rpm for 15 minutes, and the supernatant was discarded to collect the wet cells. The wet cells were added to distilled water at a concentration of 300g / L, and ultrasonically broken at 400w using an ice bath, and the cell fragmentation suspension was obtained at an interval of 1s for every 2s of fragmentation, i.e., the aconitase solution.

[0040] Example 2: Preparation of isocitric acid

[0041] 100 g of citric acid (100 g / L) was added to the initial 750 mL of water, and the pH value was adjusted to 7.2 with a 1 mol / L potassium hydroxide solution. Then, 100 mL of the aconitase solution prepared in Example 1 (wet cell concentration was 300 g / L) was added, and water was added to make the volume to 1 L before starting the reaction. During the reaction, a 1 mol / L potassium hydroxide solution was used to maintain the pH value of the reaction solution constant at 7.2, and the solution was slowly stirred at a constant temperature of 37°C for 10 h. After the reaction was completed, 1.5% activated carbon was added to the reaction solution and heated to 70°C, and the mixture was slowly stirred and kept warm for 1 h. The mixture was filtered to obtain a clear liquid, and the clear liquid was continuously rinsed with the ion exchange resin column. After the rinsing was completed, the ion exchange resin column was washed with water. After the washing was completed, the ion exchange resin column was de-eluted with 20% potassium hydroxide. The eluate was cooled to 4°C for crystallization for 15 h, and the potassium isocitrate product was obtained after solid-liquid separation and drying at 55°C for 6 h, to obtain 83.5 g of product with a purity of 99.1% and a yield of 83.5%. Potassium isocitrate was determined by high performance liquid chromatography: Chromatographic conditions: Instrument: Aglient 1200 high performance liquid chromatograph, UV detector; Chromatographic column: SB-C18, 250×4.6mm, 5μm; Flow rate: 0.8mL / min; Detection wavelength: 210nm; Mobile phase: 0.1% phosphoric acid aqueous solution: methanol = 95:5; Injection volume: 10μL; Its high performance liquid chromatogram is as follows Figure 2 shown.

[0042] Example 3

[0043] (1) Compared with Example 2, the pH was adjusted to 9, and the other conditions were the same. The final yield was only 43.0%, as shown below:

[0044] In the reaction system, citric acid, water and aconitase solution were added respectively to form a reaction mixture, and the reaction was carried out in a water bath at 37°C for 10 hours. During the reaction, the pH value of the reaction solution was controlled at 9 with potassium hydroxide solution. After the steps of activated carbon heating to adsorb impurities, filtration, ion exchange resin column washing, crystallization and drying, the potassium isocitrate product was obtained, and the calculated yield was 43.0%.

[0045] (2) Compared with Example 2, the pH of the reaction system was adjusted, and the other conditions were the same. The conditions and results are shown in Table 1.

[0046] Test Example 1 Test Example 2 Test Example 3 pH 6.0 7.0 8.0 Isocitrate yield (%) 56.9% 83.4% 64.1%

[0047] Example 4

[0048] (1) Compared with Example 2, the temperature was adjusted to 50°C, and the other conditions were the same. The final yield was only 14%, as shown below:

[0049] In the reaction system, citric acid, water and aconitase solution were added respectively to form a reaction mixture, and the reaction was carried out in a water bath at 50°C for 10 hours. During the reaction, the pH value of the reaction solution was controlled to 7.2 with a potassium hydroxide solution. After the steps of activated carbon heating to adsorb impurities, filtration, ion exchange resin column washing, crystallization and drying, the potassium isocitrate product was obtained, and the calculated yield was 14.0%.

[0050] (2) Compared with Example 2, the temperature of the reaction system was adjusted, and the other conditions were the same. The conditions and results are shown in Table 1.

[0051] Test Example 1 Test Example 2 Test Example 3 Temperature (℃) 25℃ 35℃ 45℃ Isocitrate yield (%) 56.2% 80.8% 73.7%

[0052] Example 5: Experimental device for enzymatic synthesis of isocitrate

[0053] The present invention provides a complete set of experimental devices for the enzymatic synthesis process of isocitrate described above, such as Figure 3 , 4 :

[0054] 7.5L of water and 1kg of citric acid were added to the synthesis box 10, and the pH value was adjusted to 7.2 with a 1mol / L potassium hydroxide solution. Then, 1L of aconitase prepared according to the method of Example 1 (the wet bacterial concentration was 300g / L) was added to the synthesis box 10. After adding water to make the volume to 10L, the reaction was started. During the reaction, the pH value of the reaction solution was maintained constant at 7.2 with a 1mol / L potassium hydroxide solution. The reaction solution was slowly shaken at a constant temperature of 37°C for 10 hours. When the reaction was completed, the telescopic cylinder 22 was turned on to adjust the shaking plate 27 to a suitable height during the shaking. The vibration motor 25 was turned on, and the execution end of the vibration motor 25 drove the vibration plate 24 to vibrate, and the vibration plate 24 drove the vibration plate 27 to vibrate through the connecting rod 26.

[0055] When purifying the isocitric acid solution, the telescopic cylinder 22 is opened to move the oscillation plate 27 out of the isocitric acid solution. At this time, activated carbon is placed in multiple storage boxes 28, and the total amount of activated carbon placed is 1.5% of the reaction solution. The temperature is raised to 70°C by a heater and slowly kept warm and stirred at a rate of 40 rpm for 1 hour to obtain a potassium isocitric acid solution. During stirring, the driving motor 32 drives the stirring turntable 34 to rotate, and the cylinder 33 can drive the stirring turntable 34 to rise and fall; after the reaction is completed, it is cooled to 40°C and filtered out through the filter port 13, and the filtrate is collected to obtain a potassium isocitric acid solution. The potassium isocitrate solution is discharged into the elution box 41 through the connecting pipe 43. At this time, the ion exchange resin column clamped on the clamping plate 45 adsorbs the potassium isocitrate, and the waste liquid is discharged through the drain pipe 411. After the adsorption is completed, the pneumatic cylinder 44 drives the clamping plate 45 to move downward so that the entire ion exchange resin column is located below the partition 42. 20% of the potassium hydroxide in the de-washing agent tank 46 enters the elution box 41 through the pipeline to de-wash the ion exchange resin column, and the de-washing liquid is discharged through the discharge pipe 412.

[0056] During recrystallization, the de-washing liquid was placed in a low temperature box, crystallized for 15 hours at 4°C, and after solid-liquid separation, the crystals were dried in a drying box at 55°C for 6 hours to obtain the finished potassium isocitrate, 82.0 g of product was obtained, the purity was 99.3%, and the yield was 82.0%.

[0057] It should be noted that the synthetic reaction formula of the method of the present invention is as follows Figure 5 shown. Sequence Listing <110> Changxing Pharmaceutical Co., Ltd. <120> A kind of enzymatic synthesis process of isocitrate <130> 20210316 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 2730 <212> DNA <213> Artificial sequence <400> 1 atggcaaacg agcaaaaaac tgcagcaaaa gacgttaccc aagcgagaaa aacgtttact 60 acaaatggga aaacatatca ttatactct ttaaaagcgt tagaagattc aggtatagga 120 aaggtttcga agcttcctta ttccatcaaa gttcttttag aatcagtatt gcgtcaagtt gacggcttcg ttatcaaaaa agaacacgtg gacaattgg caaaatgggg aactgccgaa 300. tcgacgttcc gttcaaaccg tctcgtgtta ttttacaaga cttcacaggg ctaccggcag tagtagatct ggcttcactg cgtaaagcaa tggcagctgt cggcggagat 360 cctgataaaa tcaaccctga aattcctgtt gatctcgtta tcgatcactc tgtacaggta 420 gataaagcgg gtacagaaga tgcattagca gtaaatatgg acttggaatt cgaaagaaat gcagagcgct acaaattttt aagctgggca aagaaagcgt ttaacaatta tcaggcagta ccgcctgcaa caggtattgt gcaccaggtc aaccttgagt tcttggcaag tgttgtccat 600 gccattgaag aagacggcga gcttgtagcu tatcctgata cgcttgtcgg aacagactca 660 cacacaacaa tgattaacgg tatcggtgtt ctcggctggg gtgtcggtgg aattgaagct gagcggga tgcttggaca gccttcttac ttcccagttc cagaggtaat cggcgcgaaa cttgtcggca agcttccaa cggaacaaca gctactgact tggcgttaa agtaacaa gtgctgcgtg aaaaaggcgt tgtcggtaaa tttgttgaat tcttcggaga aggaattgct 900 gaactgccgc ttgcagatcg cgcaacaatt gcgaatatgg ctccggaata cggtgctaca 960 tgcggattct tcccagtaga tgaagaagcg cttaactacc tgcgcctgac tggccgtgat 1020 cctgaacata ttgatgttgt tgaagcatac tgcagaagca atggcttgtt ctacactcca 1080 gatgcggaag accctcaatt tactgatgtg gttgaaattg acctgtctca aattgaagca 1140 aacttatcgg gtccaaagcg tcctcaggat cgaatcccgc tatctgctat gcaggaaacg 1200 tttaaaaagc aattagtcag ccctgcaggt aaccaaggat tcggtttaaa tgctgaggaa 1260 gaaaataag aaattaagtt taaactcctt aacggcgaag aaacagttat gaaaacgggt 1320 gcgatcgcca ttgctgcgat tacaagctgt acaaatacat caaacccata cgtgctgatc 1380 ggcgccggac tggtagcgcg taaagcggtt gagttagggc ttaaggtgcc taattacgtg 1440 aaaacgtcac ttgcaccggg ttctaaagtt gttacaggat atcttgtgaa ttcaggcctt 1500 cttccataca tgaaagagct tggctttaac ctcgttgggt acggctgtac aacatgtatc 1560 gggaactcag gtccgctttc accggaaatc gaagagcgg ttgcgaaaaa tgatcttctg 1620 attacgtctg tcctttccgg aaaccgtaac tttgaaggac gtattcaccc gcttgttaaa 1680 ggcaactatc ttgcttcacc gccgcttgtt gtggcatatg cgctggctgg aacggtaac 1740 attaacttaa aaaccgatcc aatcggtgtg ggcaaggatg gtcaaaacgt atactttaat 1800 1860 ctattccgca aagagtatga aacagtattt gatgacaaca agcgctggaa cgaaattgaa 1920 acaacagatg aagctttata taaatgggat aacgattcaa cttacatcca aaacccacca 1980 ttctttgaag agatgtctgt tgagccaggc aaggttgagc cattaaaag actgcgtgtt 2040 ctuggtaaat tcggcgattc agtcacaact gaccatattt ctcctgcgaa tgcaatcgga 2100 aaagatacgc ctgccggaaa gtatttgcaa gaaaaaggtg tttcacctcg tgactttaac 2160 2220 cgcatcaaaa accaaatcgc accgggtaca gaaggcggat ttacgacgta ctggccgact 2280 ggtgaagtaa catccatcta tgatgcatgc atgaaataca closed aaccggtctt gtcattttag caggaaaaga ctatggtatg ggatcttcac gtgactgggc tgcaaaagga acaaaccttc tcggcatcag aacggtcatt gcagagagct tcgaagaat tcacagaagc aatcttgtag gaatgggtgt gctgccgctt cagtttaaac aaggtgaaaa tcgtgataca ctcggcttaa cgggtaaaga agtcatcgag gtagatgttg atgaaacagt tcgtcctcgt gaccttgtga ctgtaagagc aatcaatga gacggcaatg taacaacttt tgaagcagtc gtccgctttg atagtgaagt tgaaattgat tactaccgcc atggcggcat ccttcaacgc gtgcttcgtg aaaaaatga gcagtcctga

Claims

1. An enzymatic synthesis process for isocitrate, characterized in that , including the following steps: Step 1: conversion of isocitric acid, in a reaction system, citric acid, water and aconitase solution are added to form a reaction mixture, and the mixture is reacted in a water bath at a constant temperature to obtain an isocitric acid reaction solution; during the reaction, aconitase catalyzes citric acid into isocitric acid; Step 2: Purification of isocitric acid: heating the isocitric acid reaction solution and adding activated carbon to absorb impurities, filtering to obtain a clear liquid, and continuously washing the ion exchange resin column with the clear liquid. After the washing is completed, the ion exchange resin column is subjected to water solid-liquid separation and dried to obtain the potassium isocitrate product; The method for obtaining the aconitase enzyme solution is as follows: using Escherichia coli BL21 (DE3) as a host strain, heterologously expressing the Cit gene encoded by aconitase from Bacillus, and obtaining an Escherichia coli engineering strain capable of catalyzing citric acid into isocitric acid; then obtaining the aconitase enzyme solution through fermentation culture and cell disruption treatment; the nucleotide sequence of the expression vector of the engineering strain contains the gene sequence as described in SEQ ID NO.

1.

2. The enzymatic synthesis process of isocitrate according to claim 1, characterized in that: In step 1, the initial reaction concentration of citric acid is 50-150 g / L.

3. The enzymatic synthesis process of isocitrate according to claim 1, characterized in that: During the reaction of step 1, the pH value is adjusted to be constant at 7.0-7.6 using 0.5-2.5 mol / L potassium hydroxide solution.

4. The enzymatic synthesis process of isocitrate according to claim 1, characterized in that: The amount of aconitase solution added in step 1 is 1-5% of the reaction system.

5. The enzymatic synthesis process of isocitrate according to claim 1, characterized in that: The catalytic reaction time in step 1 is 8-14 hours, and the reaction temperature is 30-45°C.

6. The enzymatic synthesis process of isocitrate according to claim 1, characterized in that: In step 2, the heating temperature is 55-85°C, the amount of activated carbon added is 0.5-3%, and the insulation time is 0.5-1.5h.

7. The enzymatic synthesis process of isocitrate according to claim 1, characterized in that: When the ion exchange resin column is de-eluted in step 2, the de-eluting agent is a potassium hydroxide solution with a concentration of 10-30%.

8. The enzymatic synthesis process of isocitrate according to claim 1, characterized in that: In step 2, the crystallization conditions are 0-5°C and the crystallization time is 12-18h.

9. The enzymatic synthesis process of isocitric acid according to claim 1, characterized in that: It is characterized in that The drying condition in step 2 is 50-60°C for 4-8h.

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