A method for the immobilized enzyme-catalyzed synthesis of 2,6-dihydroxybenzoic acid and the recycling of mother liquor.

Abstract

This invention relates to the field of pesticide intermediate synthesis, and discloses a method for the immobilized enzyme-catalyzed synthesis of 2,6-dihydroxybenzoic acid and the recycling of the mother liquor. The method includes: catalyzing the carboxylation reaction of resorcinol with bicarbonate in an aqueous phase using an immobilized carboxylase on MOF-74(Mn); recovering the immobilized enzyme by filtration after the reaction; crystallizing the filtrate with acid and separating the product 2,6-dihydroxybenzoic acid; extracting the remaining filtrate with an extractant, and recovering the unconverted resorcinol by distillation; and recycling the recovered immobilized enzyme and resorcinol to the next batch of reaction after replenishing with fresh material. This invention uses MOF-74(Mn) as a carrier to immobilize the carboxylase. After a single batch of carboxylation reaction, the enzyme can be directly retained and recovered using conventional solid-liquid separation methods, thus maintaining the enzyme's catalytic activity and enabling the catalyst to be recycled in subsequent batches.

Description

Technical Field

[0001] This invention relates to the field of pesticide intermediate synthesis, specifically a method for the immobilized enzyme-catalyzed synthesis of 2,6-dihydroxybenzoic acid and the recycling of mother liquor. Background Technology

[0002] 2,6-Dihydroxybenzoic acid is an important fine chemical raw material, widely used as an intermediate in the synthesis of pharmaceuticals, pesticides, and polymer materials. Traditional preparation processes mainly employ chemical synthesis, typically involving the carboxylation reaction of resorcinol with carbon dioxide or carbonates under high temperature and pressure. This chemical synthesis process requires sophisticated production equipment, and the regioselectivity of the reaction system is often unsatisfactory, easily generating multiple positional isomers as byproducts, increasing the difficulty of subsequent separation and purification. Furthermore, it generates large amounts of industrial wastewater, imposing a significant environmental treatment burden on enterprises.

[0003] To overcome the shortcomings of traditional chemical synthesis processes, enzymatic methods utilizing carboxylases to catalyze the regioselective carboxylation reaction of resorcinol with bicarbonate have gradually gained attention. Compared to chemical methods, biocatalytic systems offer advantages such as milder reaction conditions and higher product specificity. However, in existing enzymatic synthesis processes, most directly use free enzymes. Free enzymes are uniformly dissolved in the aqueous phase and are difficult to separate from the mixture after a single batch reaction, essentially making them disposable consumables and resulting in high overall production costs. Furthermore, free enzymes are highly sensitive to the external environment and are prone to structural defolding and gradual inactivation during stirring and reaction, leading to poor stability in industrial applications.

[0004] Besides the difficulty in catalyst recovery, current enzymatic synthesis processes also have significant shortcomings in material utilization. Limited by the thermodynamic equilibrium of the reaction system, resorcinol is difficult to completely convert in a single reaction. After adjusting the pH by adding acid to crystallize and filter the target product, 2,6-dihydroxybenzoic acid, a considerable amount of unreacted substrate remains in the remaining liquid mother liquor. In conventional operations, this phenol-containing mother liquor is often directly discharged as industrial wastewater, which not only results in a serious waste of raw materials but also greatly increases the difficulty of treating COD and phenolic pollutants in the wastewater. If the crystallization mother liquor is directly returned to the reactor for recycling, the inorganic salts and impurities accumulating in the system during the reaction will significantly inhibit the catalytic activity of the enzymes, leading to a decrease in product yield and purity after multiple batches of reactions.

[0005] Therefore, how to achieve stable solidification and recycling of biological enzymes, and establish an effective mechanism for the extraction and reuse of mother liquor substrates, is a technological challenge that urgently needs to be solved in this field. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for the immobilized enzyme-catalyzed synthesis of 2,6-dihydroxybenzoic acid and the recycling of the mother liquor. This method solves the problems of harsh reaction conditions, poor regioselectivity, and the generation of high-phenol-content wastewater in existing chemical methods for the synthesis of 2,6-dihydroxybenzoic acid. In contrast, traditional free biological enzyme catalysis methods face problems such as rapid catalyst deactivation, difficulty in separation and recovery, and high production costs due to the inability to utilize residual substrates in the mother liquor.

[0007] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for the immobilized enzyme-catalyzed synthesis of 2,6-dihydroxybenzoic acid and the recycling of the mother liquor, employing the following technical solution: A method for the immobilized enzyme-catalyzed synthesis of 2,6-dihydroxybenzoic acid and the recycling of the mother liquor includes the following steps: Water, MOF-74(Mn) immobilized carboxylase, resorcinol and bicarbonate were added sequentially to the reactor, and the carboxylation reaction was carried out under stirring. After the reaction was completed, the mixture was filtered, and the retained solid phase was collected as the recovered MOF-74(Mn) immobilized carboxylase, and the filtrate was collected. The pH of the filtrate was adjusted to 1 by adding acid, and the product of 2,6-dihydroxybenzoic acid was collected by filtration. Butyl acetate was added to the remaining filtrate after the product was filtered out for liquid-liquid extraction to separate the organic phase rich in unconverted resorcinol. After removing the butyl acetate by distillation, the recovered resorcinol bottom material was obtained. The collected recovered MOF-74(Mn) immobilized carboxylase and the recovered resorcinol bottom material were put back into the reactor, and fresh MOF-74(Mn) immobilized carboxylase and fresh resorcinol were added to restore the total amount of MOF-74(Mn) immobilized carboxylase and resorcinol to the initial input amount. Water and bicarbonate were added again to carry out the next batch of carboxylation reaction. The MOF-74(Mn) immobilized carboxylase is made from a MOF-74(Mn) carrier and a carboxylase with salicylate decarboxylase activity.

[0008] By adopting the above technical solution, the carboxylase is immobilized using MOF-74(Mn) and combined with liquid-liquid extraction separation technology, thus achieving the effects of improving product yield and selectivity and realizing stable multi-batch recycling of materials.

[0009] Preferably, the process parameters for the carboxylation reaction under stirring are: reaction temperature of 20-40°C and reaction time of 12-36 h.

[0010] By adopting the above technical solution, the reaction range at room temperature and pressure is defined, the irreversible denaturation of proteins caused by high temperature is avoided, the optimal catalytic activity structure of salicylate decarboxylase is maintained, and the energy consumption requirements of the production process are reduced.

[0011] Preferably, the initial input amounts of water, MOF-74(Mn) immobilized carboxylase, resorcinol, and bicarbonate are: 200-10000 parts by volume of water, 0.2-25 parts by mass of MOF-74(Mn) immobilized carboxylase, 4-600 parts by mass of resorcinol, and 2-300 parts by mass of bicarbonate.

[0012] By adopting the above technical solution, a system concentration suitable for substrate dissolution and solid-liquid mass transfer was constructed, ensuring the optimal substrate saturation required for enzyme-catalyzed reaction kinetics.

[0013] Preferably, the MOF-74(Mn) support is prepared by the following method: manganese chloride tetrahydrate and 2,5-dihydroxyterephthalic acid are added to a mixed solution of N,N-dimethylformamide and water, and after ultrasonic treatment, the solution is transferred to a reaction vessel for hydrothermal synthesis reaction; after the reaction is completed, the solution is cooled to room temperature, centrifuged, washed and vacuum dried to obtain the MOF-74(Mn) support.

[0014] By adopting the above technical solution, hydrothermal synthesis is used to promote the self-assembly of manganese ions and organic ligands, thereby constructing a porous framework structure with high crystallinity and well-developed pores, providing sufficient internal specific surface area for macromolecular enzyme protein loading.

[0015] Preferably, the process parameters for preparing the MOF-74(Mn) carrier are as follows: the initial input of raw materials is: 5-500 parts by mass of manganese chloride tetrahydrate, 1-125 parts by mass of 2,5-dihydroxyterephthalic acid, 900-45000 parts by volume of N,N-dimethylformamide, and 100-5000 parts by volume of water. The hydrothermal synthesis reaction is carried out at a temperature of 120–150°C for a duration of 12–36 h.

[0016] By adopting the above technical solution, controlling the input ratio of metal salt and ligand and the crystallization conditions, uniform growth of crystal nuclei is ensured, the generation of impurity phases is avoided, and the carrier has consistent channel morphology and structural strength.

[0017] Preferably, the MOF-74(Mn) immobilized carboxylase is prepared by the following immobilization steps: water, the MOF-74(Mn) carrier, and the carboxylase are added to the reaction system, and the loading reaction is carried out under stirring; after the reaction is completed, the reaction solution is centrifuged, washed, and freeze-dried to obtain the MOF-74(Mn) immobilized carboxylase.

[0018] By adopting the above technical solution, carboxylase molecules spontaneously embed themselves into the porous carrier in a pure water system through non-covalent interactions. Freeze-drying removes the water in the pores, preventing enzyme degradation during the shelf life.

[0019] Preferably, the process parameters for preparing MOF-74(Mn) immobilized carboxylase are as follows: the initial input of raw materials is 100-5000 parts by volume of water, 0.5-50 parts by mass of the MOF-74(Mn) carrier, and 0.05-5 parts by mass of the carboxylase; the loading reaction temperature is 20-40°C, and the time is 6-24 hours.

[0020] By adopting the above technical solution, sufficient binding time and suitable temperature are provided to ensure that the enzyme protein fully diffuses into the inner side of the carrier channel, thereby improving the overall activity of the catalyst per unit mass.

[0021] Preferably, the bicarbonate is selected from one or more of sodium bicarbonate, potassium bicarbonate, and ammonium bicarbonate.

[0022] By adopting the above technical solution, a carbon source substrate with high water solubility that can dissociate into bicarbonate and carbon dioxide molecules is provided to meet the process requirements of different reaction conditions.

[0023] Preferably, in the operation of adding acid to the filtrate to adjust the pH to 1, the added acid is hydrochloric acid; in the operation of adding butyl acetate for liquid-liquid extraction, the amount of butyl acetate added is 100 to 10,000 parts by volume.

[0024] By adopting the above technical solution, the addition of hydrochloric acid makes the system reach a specific pH value (pH=1), which removes the salt formation state of 2,6-dihydroxybenzoic acid and promotes its efficient solid-phase precipitation; butyl acetate takes into account both a moderate boiling point and excellent solubility of hydrocortisone, which greatly improves the efficiency of subsequent atmospheric distillation separation and recovery.

[0025] Preferably, the cyclic operation of re-introducing the collected and recovered MOF-74(Mn) immobilized carboxylase and the recovered resorcinol bottom material into the reactor for the next batch of reaction is performed in batches of 5.

[0026] By adopting the above technical solution, it was verified that the dual closed-loop material recovery system has good long-term operational stability, reduces the cost of the catalytic system, and has the basis for continuous engineering scale-up.

[0027] This invention provides a method for the immobilized enzyme-catalyzed synthesis of 2,6-dihydroxybenzoic acid and the recycling of the mother liquor. It has the following beneficial effects: 1. This invention uses MOF-74(Mn) as a carrier to immobilize carboxylase, thereby improving the structural stability of the free enzyme in an aqueous reaction system. Since the immobilized enzyme exists in a solid-phase form, it can be directly retained and recovered using conventional solid-liquid separation methods after a single batch of carboxylation reaction. This not only maintains the enzyme's catalytic activity but also allows the catalyst to be recycled in subsequent batches, thus reducing the high cost of using biological enzymes.

[0028] 2. This invention designs an efficient recovery mechanism for unreacted substrates in the mother liquor. After adding acid to crystallize the product and filtering, an extractant is added to the remaining filtrate for liquid-liquid extraction. Then, the solvent is removed by distillation, which can separate the unconverted resorcinol. The extracted resorcinol residue can be directly used as raw material to be added back into the next batch of reaction, which improves the comprehensive utilization rate of the substrate and reduces the discharge of phenol-containing wastewater, thus reducing the pressure on subsequent environmental treatment.

[0029] 3. This invention integrates enzymatic reaction, product crystallization, and dual recycling of catalyst and substrate into a complete closed-loop production process. It is carried out under mild conditions, with simple process steps, avoiding the cumbersome separation and purification steps in traditional synthesis methods. While ensuring high yield and purity of the target product 2,6-dihydroxybenzoic acid, it exhibits good process stability and reproducibility, and is feasible for industrial-scale production. Attached Figure Description

[0030] Figure 1 This is a bar chart comparing the product yield and purity after repeated use in various embodiments of the present invention. Figure 2 This is a line graph showing the changes in product yield and purity during multiple cycles of this invention. Figure 3 The bar chart shows the comparison of product yield and purity between the embodiments of the present invention and the comparative examples under different catalytic systems. Figure 4 This is a bar chart comparing the composition and selectivity distribution of products in the embodiments and comparative examples of the present invention under different process conditions; Figure 5 Schematic diagram of the synthetic route of this invention Figure 6 High performance liquid chromatogram of 2,6-dihydroxybenzoic acid synthesized in Example 1. Detailed Implementation

[0031] The technical solutions of the present invention will be clearly and completely described below with reference to embodiments, comparative examples, and test examples. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] In the following preparation examples and embodiments, the conversion relationship between mass parts and volume parts is as follows: when mass parts are expressed in grams and volume parts are expressed in milliliters, the ratio of the two values ​​is 1:1.

[0033] Preparation Examples 1-4: Preparation Example 1: This preparation example provides a method for preparing MOF-74(Mn) immobilized carboxylase, including the following steps: (1) Preparation of MOF-74(Mn): At room temperature, 10 parts by mass (10 g) of manganese chloride tetrahydrate was dissolved in a mixed solution of 900 parts by volume (900 mL) of N,N-dimethylformamide and 100 parts by volume (100 mL) of water and ultrasonically treated for 30 min. Then, 2.5 parts by mass (2.5 g) of 2,5-dihydroxyterephthalic acid was added to the mixed solution, and ultrasonic treatment was continued for 30 min. The mixed solution was then transferred to a polytetrafluoroethylene-lined reactor and subjected to hydrothermal synthesis at 135 °C for 24 h. After the reaction was completed, the temperature was lowered to room temperature, and 3 parts by mass (3 g) of MOF-74(Mn) was obtained by centrifugation, washing, and vacuum drying for 36 h.

[0034] (2) Preparation of immobilized carboxylase: In a reaction vessel, 100 parts by volume (100 mL) of water, 1 part by mass (1 g) of the MOF-74 (Mn) prepared above, and 0.1 part by mass (0.1 g) of carboxylase (with salicylate decarboxylase Sdc activity) were added sequentially, and the mixture was stirred at 30 °C for 12 h for loading reaction. After stirring, the reaction solution was centrifuged, washed, and freeze-dried for 24 h to obtain 1 part by mass (1 g) of MOF-74 (Mn) immobilized carboxylase.

[0035] Preparation Example 2: This preparation example provides a method for preparing MOF-74(Mn) immobilized carboxylase, including the following steps: (1) Preparation of MOF-74(Mn): At room temperature, 5 parts by mass (5g) of manganese chloride tetrahydrate were dissolved in a mixed solution of 900 parts by volume (900mL) of N,N-dimethylformamide and 100 parts by volume (100mL) of water and ultrasonically treated for 30min. Then, 1 part by mass (1g) of 2,5-dihydroxyterephthalic acid was added to the mixed solution and ultrasonic treatment was continued for 30min. The mixed solution was then transferred to a polytetrafluoroethylene-lined reactor and hydrothermal synthesis reaction was carried out at 120℃ for 12h. After the reaction was completed, the temperature was lowered to room temperature, and 1 part by mass (1g) of MOF-74(Mn) was obtained by centrifugation, washing, and vacuum drying for 24h.

[0036] (2) Preparation of immobilized carboxylase: In a reaction vessel, 100 parts by volume (100 mL) of water, 0.5 parts by mass (0.5 g) of the MOF-74 (Mn) prepared above, and 0.05 parts by mass (0.05 g) of carboxylase (with salicylate decarboxylase Sdc activity) were added sequentially. The mixture was stirred at 20 °C for 6 h for loading reaction. After stirring, the reaction solution was centrifuged, washed, and freeze-dried for 12 h to obtain 0.4 parts by mass (0.4 g) of MOF-74 (Mn) immobilized carboxylase.

[0037] Preparation Example 3: This preparation example provides a method for preparing MOF-74(Mn) immobilized carboxylase, including the following steps: (1) Preparation of MOF-74(Mn): At room temperature, 20 parts by mass (20 g) of manganese chloride tetrahydrate was dissolved in a mixed solution of 900 parts by volume (900 mL) of N,N-dimethylformamide and 100 parts by volume (100 mL) of water and ultrasonically treated for 30 min. Then, 5 parts by mass (5 g) of 2,5-dihydroxyterephthalic acid was added to the mixed solution, and ultrasonic treatment was continued for 30 min. The mixed solution was then transferred to a polytetrafluoroethylene-lined reactor and subjected to hydrothermal synthesis at 150 °C for 36 h. After the reaction was completed, the temperature was lowered to room temperature, and 6.2 parts by mass (6.2 g) of MOF-74(Mn) was obtained by centrifugation, washing, and vacuum drying for 48 h.

[0038] (2) Preparation of immobilized carboxylase: In a reaction vessel, 100 parts by volume (100 mL) of water, 2 parts by mass (2 g) of the MOF-74 (Mn) prepared above, and 0.2 parts by mass (0.2 g) of carboxylase (with salicylate decarboxylase Sdc activity) were added sequentially, and the mixture was stirred at 40 °C for 24 h for loading reaction. After stirring, the reaction solution was centrifuged, washed, and freeze-dried for 36 h to obtain 2 parts by mass (2 g) of MOF-74 (Mn) immobilized carboxylase.

[0039] Preparation Example 4: This preparation example provides a method for preparing MOF-74(Mn) immobilized carboxylase, including the following steps: (1) Preparation of MOF-74(Mn): At room temperature, 500 parts by mass (500 g) of manganese chloride tetrahydrate was dissolved in a mixed solution of 45,000 parts by volume (45 L) of N,N-dimethylformamide and 5,000 parts by volume (5 L) of water and ultrasonically treated for 30 min. Then, 125 parts by mass (125 g) of 2,5-dihydroxyterephthalic acid was added to the mixed solution, and ultrasonic treatment was continued for another 30 min. The mixed solution was then transferred to a polytetrafluoroethylene-lined reactor and subjected to a hydrothermal synthesis reaction at 135 °C for 24 h. After the reaction was completed, the temperature was lowered to room temperature, and 147 parts by mass (147 g) of MOF-74(Mn) was obtained by centrifugation, washing, and vacuum drying for 36 h.

[0040] (2) Preparation of immobilized carboxylase: In a reaction vessel, 5000 parts by volume (5L) of water, 50 parts by mass (50g) of the MOF-74 (Mn) prepared above, and 5 parts by mass (5g) of carboxylase (with salicylate decarboxylase Sdc activity) were added sequentially, and the mixture was stirred at 30℃ for 12h for loading reaction. After stirring, the reaction solution was centrifuged, washed, and freeze-dried for 24h to obtain 48 parts by mass (48g) of MOF-74 (Mn) immobilized carboxylase.

[0041] Examples 1-4: The schematic diagram of the synthetic route of this invention is shown below. Figure 5 As shown. Example 1:

[0042] This embodiment provides a method for the immobilized enzyme-catalyzed synthesis of 2,6-dihydroxybenzoic acid and the recycling of the mother liquor, including the following steps: In a reaction vessel, 200 parts by volume (200 mL) of water, 0.5 parts by weight (0.5 g) of the MOF-74(Mn) immobilized carboxylase obtained in Preparation Example 1, 12 parts by weight (12 g) of resorcinol, and 6 parts by weight (6 g) of potassium bicarbonate (a type of bicarbonate) were added sequentially. The reaction was carried out by stirring at 30 °C for 24 h. After the reaction was completed, the mixture was filtered to recover the MOF-74(Mn) immobilized carboxylase, which was then collected for later use.

[0043] Hydrochloric acid was added to the filtrate to adjust the pH to 1, and the product 2,6-dihydroxybenzoic acid was obtained by filtration. 200 parts by volume (200 mL) of butyl acetate was added to all the filtrate collected after product filtration to extract unreacted resorcinol. After extraction, butyl acetate was recovered by distillation, and the residue at the bottom of the distillate was resorcinol, which was collected for later use.

[0044] When recycled for the next batch of reaction, the recovered MOF-74(Mn) immobilized carboxylase and resorcinol from the reactor bottom were added to the reactor, and fresh MOF-74(Mn) immobilized carboxylase and fresh resorcinol were added to restore the total amount of immobilized enzyme to 0.5 parts by mass (0.5 g) and the total amount of resorcinol to 12 parts by mass (12 g). Then, 200 parts by volume (200 mL) of water and 6 parts by mass (6 g) of potassium bicarbonate were added again for the next batch of carboxylation reaction. After 5 cycles, the yield of 2,6-dihydroxybenzoic acid was measured to be 95.71%, and the purity was 99.97%. Its high-performance liquid chromatography chromatogram is shown below. Figure 6 As shown. Example 2:

[0045] This embodiment provides a method for the immobilized enzyme-catalyzed synthesis of 2,6-dihydroxybenzoic acid and the recycling of the mother liquor, including the following steps: In a reaction vessel, 200 parts by volume (200 mL) of water, 0.2 parts by mass (0.2 g) of the MOF-74(Mn) immobilized carboxylase obtained in Preparation Example 2, 4 parts by mass (4 g) of resorcinol, and 2 parts by mass (2 g) of sodium bicarbonate (a type of bicarbonate) were added sequentially. The reaction was carried out by stirring at 20 °C for 12 h. After the reaction was completed, the mixture was filtered to recover the MOF-74(Mn) immobilized carboxylase, which was then collected for later use.

[0046] Hydrochloric acid was added to the filtrate to adjust the pH to 1, and the product 2,6-dihydroxybenzoic acid was obtained by filtration. 100 parts by volume (100 mL) of butyl acetate was added to all the filtrate collected after product filtration to extract unreacted resorcinol. After extraction, butyl acetate was recovered by distillation, and the residue at the bottom of the vessel was resorcinol, which was collected for later use.

[0047] When recycled for the next batch of reaction, the recovered MOF-74(Mn) immobilized carboxylase and resorcinol from the reactor bottom were added to the reactor, and fresh MOF-74(Mn) immobilized carboxylase and fresh resorcinol were added to restore the total amount of immobilized enzyme to 0.2 parts by mass (0.2 g) and the total amount of resorcinol to 4 parts by mass (4 g). Then, 200 parts by volume (200 mL) of water and 2 parts by mass (2 g) of sodium bicarbonate were added again for the next batch of carboxylation reaction. After 5 cycles, the yield of 2,6-dihydroxybenzoic acid was measured to be 93.65%, and the purity was 98.17%. Example 3:

[0048] This embodiment provides a method for the immobilized enzyme-catalyzed synthesis of 2,6-dihydroxybenzoic acid and the recycling of the mother liquor, including the following steps: In a reaction vessel, 200 parts by volume (200 mL) of water, 1 part by mass (1 g) of the MOF-74(Mn) immobilized carboxylase obtained in Preparation Example 3, 20 parts by mass (20 g) of resorcinol, and 10 parts by mass (10 g) of ammonium bicarbonate (a type of bicarbonate) were added sequentially. The reaction was carried out by stirring at 40 °C for 36 h. After the reaction was completed, the mixture was filtered to recover the MOF-74(Mn) immobilized carboxylase, which was then collected for later use.

[0049] Hydrochloric acid was added to the filtrate to adjust the pH to 1, and the product 2,6-dihydroxybenzoic acid was obtained by filtration. 400 parts by volume (400 mL) of butyl acetate was added to all the filtrate collected after product filtration to extract unreacted resorcinol. After extraction, butyl acetate was recovered by distillation, and the residue at the bottom of the distillate was resorcinol, which was collected for later use.

[0050] When recycled for the next batch of reaction, the recovered MOF-74(Mn) immobilized carboxylase and resorcinol from the reactor bottom were added to the reactor, and fresh MOF-74(Mn) immobilized carboxylase and fresh resorcinol were added to restore the total amount of immobilized enzyme to 1 part by mass (1 g) and the total amount of resorcinol to 20 parts by mass (20 g). Then, 200 parts by volume (200 mL) of water and 10 parts by mass (10 g) of ammonium bicarbonate were added again for the next batch of carboxylation reaction. After 5 cycles, the yield of 2,6-dihydroxybenzoic acid was measured to be 98.28%, and the purity was 99.45%. Example 4:

[0051] This embodiment provides a method for the immobilized enzyme-catalyzed synthesis of 2,6-dihydroxybenzoic acid and the recycling of the mother liquor, including the following steps: In a reaction vessel, 10,000 parts by volume (10 L) of water, 25 parts by weight (25 g) of the MOF-74(Mn) immobilized carboxylase obtained in Preparation Example 4, 600 parts by weight (600 g) of resorcinol, and 300 parts by weight (300 g) of potassium bicarbonate (a type of bicarbonate) were added sequentially. The reaction was carried out by stirring at 30 °C for 24 h. After the reaction was completed, the mixture was filtered to recover the MOF-74(Mn) immobilized carboxylase, which was then collected for later use.

[0052] Hydrochloric acid was added to the filtrate to adjust the pH to 1, and the product 2,6-dihydroxybenzoic acid was obtained by filtration. 10,000 parts by volume (10 L) of butyl acetate was added to all the filtrate collected after product filtration to extract unreacted resorcinol. After extraction, butyl acetate was recovered by distillation, and the residue at the bottom of the vessel was resorcinol, which was collected for later use.

[0053] When recycled for the next batch of reaction, the recovered MOF-74(Mn) immobilized carboxylase and resorcinol from the reactor bottom were added to the reactor, and fresh MOF-74(Mn) immobilized carboxylase and fresh resorcinol were added to restore the total amount of immobilized enzyme to 25 parts by mass (25 g) and the total amount of resorcinol to 600 parts by mass (600 g). Then, 10,000 parts by volume (10 L) of water and 300 parts by mass (300 g) of potassium bicarbonate were added again for the next batch of carboxylation reaction. After 5 cycles, the yield of 2,6-dihydroxybenzoic acid was measured to be 96.79%, and the purity was 98.23%.

[0054] Comparative Examples 1-4: Comparative Example 1: Compared with Example 1, the difference is that MOF-74(Mn) is not used as a carrier, and an equal amount of free carboxylase is added directly; otherwise, they are the same.

[0055] Comparative Example 2: Compared with Example 1, the difference is that conventional macroporous resin was used instead of MOF-74(Mn) for enzyme immobilization, otherwise the same.

[0056] Comparative Example 3: Compared with Example 1, the difference is that in the preparation steps, manganese chloride tetrahydrate is replaced with an equimolar amount of zinc chloride to prepare MOF-74(Zn) with zinc as the metal center to immobilize the carboxylase. All other steps are the same.

[0057] Comparative Example 4: Compared with Example 1, the difference is that no immobilized enzyme or free enzyme is added, and the reaction system is placed under high temperature and high pressure conditions of 100°C and 5MPa to carry out a conventional chemical carboxylation reaction. All other aspects are the same.

[0058] Test Examples 1-4: Test Example 1: Efficient Synthesis and Purity Verification of the Target Product This test case is used to verify the yield and purity of the target product 2,6-dihydroxybenzoic acid prepared by the processes constructed in Examples 1 to 4.

[0059] Accurately weigh 10.00 mg of dried 2,6-dihydroxybenzoic acid product from the final batch after five cycles of Examples 1 to 4, place it in a 10 mL volumetric flask, dissolve it in methanol and dilute to the mark, filter it through a 0.22 μm organic filter membrane to obtain the sample solution to be tested.

[0060] 2,6-Dihydroxybenzoic acid standard solutions with different concentration gradients were prepared, with concentrations of 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL and 5.0 mg / mL, respectively.

[0061] The determination was performed using a high-performance liquid chromatograph (Agilent 1260). The chromatographic conditions were set as follows: ZORBAX SB-C8 column (4.6 × 250 mm, 5 μm); mobile phase: a mixture of 0.1% formic acid aqueous solution and methanol; injection volume: 5 μL; flow rate: 1.0 mL / min; detection wavelength: 230 nm; column temperature: 40 °C.

[0062] A standard curve was plotted by sequentially injecting standard solutions. Subsequently, each test sample solution was analyzed, and the chromatographic peak area was recorded. The product purity was calculated using the area normalization method, and the yield was calculated by combining the actual collected product quality with the theoretical output quality.

[0063] Table 1. Yield and purity data of products after 5 cycles in each example

[0064] according to Figure 1 According to the data in Table 1, under different reaction parameters and preparation scales, the yield of the target product 2,6-dihydroxybenzoic acid in Examples 1 to 4 ranged from 93.65% to 98.28%, and the chromatographic purity remained between 98.17% and 99.97%. Figure 1 The data clearly shows that under different process conditions and in Example 4, which was scaled up, the yield and purity data bars remained high with small fluctuations between groups.

[0065] Under normal conditions, salicylate decarboxylase mainly exhibits decarboxylation activity in degrading 2,6-dihydroxybenzoic acid into resorcinol and carbon dioxide. Figure 1 The high yield conversion data from the various embodiments indicate that the thermodynamic equilibrium within the reaction system shifted towards synthesis. The pore structure of the MOF-74(Mn) support exhibits physical enrichment properties for carbon dioxide, increasing the local substrate concentration within the enzyme catalytic microenvironment. Simultaneously, the high-density distribution of coordinating unsaturated manganese metal sites in the support backbone synergistically interacts with the active site of the enzyme molecule. Manganese ions participate in regulating the local conformation and electron transfer processes of the enzyme protein, lowering the activation energy of the reverse carboxylation reaction, and activating and dominating the carboxylation synthesis pathway of this enzyme.

[0066] Figure 1The purity data reflects the regioselectivity of the catalytic system. In traditional chemical synthesis, the carboxylation of resorcinol tends to occur at multiple sites, leading to the formation of isomer byproducts such as 2,4-dihydroxybenzoic acid. In this scheme, the immobilized carboxylase combines the enzyme's substrate specificity with the three-dimensional steric hindrance effect of the MOF-74(Mn) micropores, restricting the binding orientation of the substrate molecule at the catalytic site and suppressing carboxylation side reactions at non-target sites. Example 4 in Figure 1 The yield and purity of the enzyme were similar to those of Example 1, indicating that the immobilized enzyme maintained uniform mass transfer efficiency and catalytic activity in the scale-up system, verifying the feasibility of continuous preparation at the hundred-gram scale and above.

[0067] Test Example 2: Stability Assessment of Dual Circulation of Mother Liquor This test example uses the process parameters of Example 1 as a representative to determine the catalyst activity and the stability of the reaction system during multiple solid-liquid separations and material recycling processes.

[0068] The mixture after the initial batch reaction in Example 1 was collected, and solid-liquid separation was performed by filtration. The solid phase on the filter paper was retained as the recovered MOF-74(Mn) immobilized carboxylase. After removing the surface moisture in a vacuum drying oven, its dry weight was recorded.

[0069] Hydrochloric acid was added dropwise to the mother liquor to adjust the pH to 1. The precipitated 2,6-dihydroxybenzoic acid crystals were filtered, dried, weighed, and their purity was determined using a high-performance liquid chromatograph (Agilent 1260). The yield of the current batch was calculated based on the amount of substrate consumed.

[0070] Collect all the filtrate after the product is precipitated, add an equal volume of butyl acetate for liquid-liquid extraction, separate the organic phase and then perform atmospheric distillation to recover butyl acetate. Collect the unreacted resorcinol residue at the bottom of the distillation vessel and weigh it.

[0071] The recovered MOF-74(Mn) immobilized carboxylase and recovered resorcinol were added to the reaction system, and the mass loss of the two compared with the initial input amount was measured. The corresponding amounts of fresh MOF-74(Mn) immobilized carboxylase and fresh resorcinol were added to restore the total amount of immobilized enzyme in the reactor to 0.5g and the total amount of resorcinol to 12g.

[0072] Add 200mL of water and 6g of potassium bicarbonate to start the next batch reaction. Repeat the above procedure for 5 consecutive cycles, recording the material recovery and replenishment data for each batch, as well as the physicochemical properties of the final product.

[0073] Table 2. Material recovery and replenishment records and product indicator test results for each cycle batch in Example 1

[0074] Note: "-" indicates not applicable (this batch is the first reaction and there is no recovered material).

[0075] according to Figure 2 According to the data in Table 2, in the initial batch and the subsequent five consecutive cycles of Example 1, the product yield of each batch ranged from 95.53% to 96.31%, and the chromatographic purity was maintained above 99.91%. Figure 2 The broken line trend shows that after the reaction system underwent multiple solid-liquid separation, acid-base adjustment and material replenishment operations, the yield curve and purity curve remained stable, and there was no decrease in conversion rate and selectivity.

[0076] The data reflect the physical confinement effect of the rigid porous framework structure of the MOF-74(Mn) support on the internally loaded salicylate decarboxylase. This physical confinement reduces the probability of conformational unfolding and active site inactivation of the salicylate decarboxylase protein during mechanical stirring and pH adjustment. The material recovery records in Table 2 show that the mass loss of the immobilized enzyme was controlled between 0.02 g and 0.05 g per cycle. The interaction forces between the support framework and the salicylate decarboxylase molecules limit the catalyst from detaching into the aqueous solution.

[0077] Unconverted resorcinol in the filtrate was separated by butyl acetate liquid-liquid extraction and atmospheric distillation, and then returned to the reaction system in a closed loop. No impurities that inhibit enzyme catalytic activity were introduced into the reaction vessel during the extraction and separation process. Figure 2 No fluctuations were observed in the product purity curve. Before each batch reaction, fresh immobilized enzyme and resorcinol were replenished based on material analysis data to restore the initial material ratio inside the reactor and maintain the substrate concentration gradient between the immobilized carrier pores and the external aqueous solution. The physical reuse of the immobilized enzyme across multiple batches amortized the catalyst cost, and the closed-loop recovery of unreacted resorcinol reduced substrate consumption. These data demonstrate the engineering basis of the continuous operation process.

[0078] Test Example 3: Comparative Test of Synergistic Effects between Catalytic Microenvironment and Specific Metal Sites This test case is used to verify the synergistic mechanism of the catalyst support microenvironment and specific central metal ions in driving the reverse carboxylation reaction.

[0079] The MOF-74(Mn) immobilized carboxylase prepared in Example 1, the free carboxylase in Comparative Example 1, the macroporous resin immobilized carboxylase in Comparative Example 2, and the MOF-74(Zn) immobilized carboxylase in Comparative Example 3 were taken respectively. Based on the carrier loading rate calculation data, the absolute mass of salicylate decarboxylase in the input materials of each group was controlled to be equal.

[0080] In four separate reactors, 200 mL of water, the corresponding catalyst group, 12.08 g of resorcinol, and 6.05 g of potassium bicarbonate were added sequentially. Mechanical stirring was started, with the speed controlled at 200 rpm, and the reaction system temperature controlled at 30°C. The reactors were run continuously for 24 hours.

[0081] After the reaction was completed, the reaction mixtures of Example 1, Comparative Example 2, and Comparative Example 3 were filtered to separate the solid-phase catalyst. For the free enzyme system of Comparative Example 1, an ultrafiltration device was used to retain enzyme protein molecules in the aqueous phase, and the clear liquid mother liquor of each group was collected.

[0082] Add 3 mol / L hydrochloric acid solution dropwise to the obtained mother liquor, monitor the pH in real time with a pH meter, adjust the pH of the system to 1, and let it stand at room temperature for 2 hours to allow the product to crystallize out.

[0083] The precipitates from each group were collected by filtration and the moisture was removed using a vacuum drying oven. The mass of the dried solid product was accurately weighed, and the quantitative conversion data and chromatographic purity were determined using high-performance liquid chromatography (HPLC).

[0084] Table 3. Product test data of the catalytic systems in Example 1 and each comparative example

[0085] according to Figure 3 According to the data in Table 3, the product yield of Example 1 was 95.82%, and the yield of the free carboxylase system in Comparative Example 1 was 7.43%. Figure 3 The columnar contrast analysis showed that the conversion rate of the free enzyme system was extremely low. Salicylate decarboxylase, in its free state, primarily exhibits a decarboxylation degradation pathway. Monomeric enzyme molecules lack the mechanism for retaining and enriching carbon dioxide or bicarbonate substrates in an aqueous environment; the reaction kinetics are constrained by the energy barrier of reverse carboxylation synthesis, thus hindering the conversion of the target product.

[0086] Comparative Example 2 used macroporous resin as the immobilization carrier. Figure 3 The product yield was 12.15%. The physical confinement environment provided by the macroporous resin reduced the effect of solution mechanical shearing on the enzyme molecule conformation, resulting in a slight increase in yield. The polymer resin lacked metal catalytic sites and specific gas loading channels, failing to construct a local catalytic microenvironment that promoted carbon capture, and the thermodynamic equilibrium within the reaction system did not shift towards carboxylation synthesis.

[0087] Comparative Example 3 used a MOF-74 support with the central metal replaced by zinc, and the product yield was 28.67%. The zinc-based porous material retained its framework topology and carbon dioxide enrichment capacity, improving the local substrate concentration within the pore microenvironment. Figure 3The yield column height of this method is superior to that of the macroporous resin system. The difference in yield data between Example 1 and Comparative Example 3 reflects the synergistic activation effect of specific metal central sites. The coordinatingly unsaturated manganese metal sites in the MOF-74(Mn) framework possess electron configuration characteristics adapted to this enzymatic reaction pathway. Manganese ions act as inorganic metal cofactors, participating in the electron transfer process between substrate molecules and the enzyme active site. The test results indicate that the basic substrate concentration effect of porous materials needs to be combined with the catalytic activation mechanism of specific metal sites to drive the efficient conversion of this reverse enzymatic reaction.

[0088] Test Example 4: Comparative Test of High Selectivity and Byproduct Inhibition at Room Temperature and Pressure This test case is used to verify the regioselectivity of the proposed method under ambient temperature and pressure enzymatic conditions, as well as its inhibitory effect on isomer byproducts, and to compare it with the traditional high temperature and high pressure chemical carboxylation method.

[0089] Take the product powder that was precipitated by acid adjustment and vacuum dried after the reaction of Example 1; simultaneously take the product powder that was precipitated and dried after the chemical reaction of Comparative Example 4 at 100°C and 5MPa, and then subjected to the same acid adjustment and standing steps.

[0090] Accurately weigh 10.05 mg of each of the two solid samples and place them in a 10 mL volumetric flask. Add methanol to dissolve the samples with ultrasonic assistance and make up to volume. Filter the solutions through a 0.22 μm organic phase microporous membrane to prepare the two test solutions.

[0091] Compositional analysis was performed using high-performance liquid chromatography (HPLC). A C18 reversed-phase column (4.6 × 250 mm, 5 μm) was used, and the mobile phase was set as an isocratic elution system of methanol and 0.1% phosphoric acid aqueous solution. The detection wavelength was 230 nm.

[0092] Two test solutions were injected and analyzed separately, and the chromatograms were recorded. The peak positions of the main product and by-products were determined by comparing the retention times of 2,6-dihydroxybenzoic acid and 2,4-dihydroxybenzoic acid standards. The mass percentage content of each component in the solid product was calculated using the peak area normalization method.

[0093] Table 4. Product composition and selectivity distribution data of Example 1 and Comparative Example 4

[0094] according to Figure 4Based on the data in Table 4, in the solid product of Example 1 under normal pressure and 30°C, the content of the target product 2,6-dihydroxybenzoic acid reached 99.93%, and the content of the isomer byproduct 2,4-dihydroxybenzoic acid was 0.04%. In the chemically produced product of Comparative Example 4 under 100°C and 5 MPa conditions, the content of 2,6-dihydroxybenzoic acid was 42.87%, and the content of 2,4-dihydroxybenzoic acid was 51.35%. Figure 4 The stacked bar distribution reflects the even distribution of main and by-products in the product of Comparative Example 4, as well as the high selectivity of the product of Example 1.

[0095] Traditional high-temperature, high-pressure chemical carboxylation reactions lack regioselectivity. The two hydroxyl groups on the resorcinol benzene ring exert electron activation on their ortho and para positions. In the high-temperature, high-pressure system, the electrophilic attack of carbon dioxide molecules is kinetically controlled. The steric hindrance of the carbon atom at position 4 of resorcinol is smaller than that of the carbon atom at position 2 between the two hydroxyl groups, making carboxylation at position 4 more likely, resulting in a high proportion of 2,4-dihydroxybenzoic acid in the product. The coexistence of isomers increases the operational difficulty of subsequent crystallization and separation processes.

[0096] Figure 4 Data from Example 1 validated the steric confinement effect of the enzymatic system. In the immobilized system, the active pocket of salicylate decarboxylase possesses a specific structure targeting the substrate molecule, and the porous rigid framework of MOF-74(Mn) constructs a physically confined space around the enzyme molecule. When the resorcinol substrate diffuses into the catalytic active site, its spatial binding orientation is dually constrained by the carrier pore size and the amino acid residues of the enzyme protein. Carbon dioxide molecules are confined within specific channels and can only attack the carbon atom at position 2 of resorcinol. The steric hindrance effect blocks the reaction pathway of substrate carboxylation at position 4, inhibiting the formation of 2,4-dihydroxybenzoic acid. The reaction achieves the directional conversion of the substrate under ambient temperature and pressure conditions, reducing the production system's dependence on high-pressure equipment and simplifying the mixture separation process.

Claims

1. A method for the immobilized enzyme-catalyzed synthesis of 2,6-dihydroxybenzoic acid and the recycling of the mother liquor, characterized in that, Includes the following steps: Water, MOF-74(Mn) immobilized carboxylase, resorcinol and bicarbonate were added sequentially to the reactor, and the carboxylation reaction was carried out under stirring. After the reaction was completed, the mixture was filtered, and the retained solid phase was collected as the recovered MOF-74(Mn) immobilized carboxylase, and the filtrate was collected. The pH of the filtrate was adjusted to 1 by adding acid, and the product of 2,6-dihydroxybenzoic acid was collected by filtration. Butyl acetate was added to the remaining filtrate after the product was filtered out for liquid-liquid extraction to separate the organic phase rich in unconverted resorcinol. After removing the butyl acetate by distillation, the recovered resorcinol bottom material was obtained. The collected recovered MOF-74(Mn) immobilized carboxylase and the recovered resorcinol bottom material were put back into the reactor, and fresh MOF-74(Mn) immobilized carboxylase and fresh resorcinol were added to restore the total amount of MOF-74(Mn) immobilized carboxylase and resorcinol to the initial input amount. Water and bicarbonate were added again to carry out the next batch of carboxylation reaction. The MOF-74(Mn) immobilized carboxylase is made from a MOF-74(Mn) carrier and a carboxylase with salicylate decarboxylase activity.

2. The method according to claim 1, characterized in that, The process parameters for the carboxylation reaction under stirring are: reaction temperature of 20-40℃ and reaction time of 12-36h.

3. The method according to claim 1, characterized in that, The initial input amounts of water, MOF-74(Mn) immobilized carboxylase, resorcinol, and bicarbonate are as follows: 200–10,000 parts by volume of water, 0.2–25 parts by mass of MOF-74(Mn) immobilized carboxylase, 4–600 parts by mass of resorcinol, and 2–300 parts by mass of bicarbonate.

4. The method according to claim 1, characterized in that, The MOF-74(Mn) support was prepared by the following method: Manganese chloride tetrahydrate and 2,5-dihydroxyterephthalic acid were added to a mixed solution of N,N-dimethylformamide and water. After ultrasonic treatment, the solution was transferred to a reaction vessel for hydrothermal synthesis. After the reaction was completed, the temperature was lowered to room temperature, and the MOF-74(Mn) support was obtained by centrifugation, washing and vacuum drying.

5. The method according to claim 1, characterized in that, The MOF-74(Mn) immobilized carboxylase was prepared via the following immobilization steps: Water, the MOF-74(Mn) carrier, and the carboxylase were added to the reaction system, and the loading reaction was carried out under stirring. After the reaction was completed, the reaction solution was centrifuged, washed and freeze-dried to obtain the MOF-74(Mn) immobilized carboxylase.

6. The method according to claim 1, characterized in that, The bicarbonate is selected from one or more of sodium bicarbonate, potassium bicarbonate, and ammonium bicarbonate.

7. The method according to claim 4, characterized in that, The process parameters for preparing the MOF-74(Mn) support are within the following range: The initial input of raw materials is: 5-500 parts by weight of manganese chloride tetrahydrate, 1-125 parts by weight of 2,5-dihydroxyterephthalic acid, 900-45000 parts by volume of N,N-dimethylformamide, and 100-5000 parts by volume of water. The hydrothermal synthesis reaction is carried out at a temperature of 120–150°C for a duration of 12–36 h.

8. The method according to claim 5, characterized in that, The process parameters for preparing MOF-74(Mn) immobilized carboxylase are within the following range: The initial input of raw materials is: 100-5000 parts by volume of water, 0.5-50 parts by mass of the MOF-74(Mn) carrier, and 0.05-5 parts by mass of the carboxylase; The loading reaction is carried out at a temperature of 20–40°C for 6–24 hours.

9. The method according to claim 1, characterized in that, In the operation of adding acid to the filtrate to adjust the pH to 1, the acid added is hydrochloric acid; In the operation of adding butyl acetate for liquid-liquid extraction, the amount of butyl acetate added is 100 to 10,000 parts by volume.

10. The method according to claim 1, characterized in that, The cyclic operation of re-introducing the collected and recovered MOF-74(Mn) immobilized carboxylase and the recovered resorcinol substrate into the reactor for the next batch of reaction is repeated 5 times.

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