Method for preparing acrylamide by constructing resin-MOFs-enzyme system biological method

Abstract

The invention discloses a biological method for preparing acrylamide by constructing a resin-MOFs-enzyme system. The preparation materials of the resin-MOFs-enzyme system comprise pure water, resin, metal salt, ligand, sodium hydroxide and nitrile hydratase. According to the synthetic method, MOF is loaded on resin, then nitrile hydratase is uniformly fixed to pore channels and the surface of the resin through electrostatic interaction and adsorption, a resin-MOFs-enzyme system is formed, the stability of enzyme can be improved, and acrylonitrile can be better converted into acrylamide.

Description

[0001] This application relates to the technical field of biocatalytic production of acrylamide, and specifically relates to a method for preparing acrylamide by constructing a resin-MOFs-enzyme system through a biological method. Background Art

[0002] Acrylamide is an important organic chemical raw material, widely used in fields such as oil extraction, papermaking, textile, and sewage treatment. The traditional production method of acrylamide is mainly the chemical method, which is prepared by the hydration reaction of acrylonitrile. However, the chemical method has some disadvantages, such as harsh reaction conditions, serious environmental pollution, low product purity, etc. With the development of biotechnology, the biological method for preparing acrylamide has gradually become a research hotspot. The biological method has the advantages of mild reaction conditions, environmental friendliness, high product purity, etc., which conforms to the development trend of green chemistry. At present, some microorganisms have been found to be able to produce nitrile hydratase, which can catalyze the hydration reaction of acrylonitrile to produce acrylamide. The key to the biological method for preparing acrylamide lies in screening highly efficient nitrile hydratase-producing bacteria and optimizing their fermentation conditions and reaction processes. In addition, problems such as the stability and reusability of biocatalysts need to be solved to improve production efficiency and reduce costs.

[0003] This patent aims to provide a method for preparing acrylamide by constructing a resin-MOFs-enzyme system through a biological method. By loading nitrile hydratase onto the resin-MOFs system, high-efficiency and green production of acrylamide can be achieved. This method has broad application prospects and will provide new technical support for the sustainable development of the acrylamide industry. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to improve the stability and catalytic ability of nitrile hydratase.

[0005] The present invention provides a method for preparing acrylamide by constructing a resin-MOFs-enzyme system through a biological method. The preparation method includes the following steps: (1) Transfer 1.5 - 2.5 parts of resin, 0.2 - 0.6 parts of ligand, and 0 - 0.5 parts of sodium hydroxide to pure water by mass fraction in sequence. After slowly stirring for 1 - 2 h, wash with pure water 3 - 5 times until neutral; dissolve 0.3 - 0.8 parts of metal salt in 25 - 35 parts of solvent, then transfer the washed resin above to the metal salt solution, stir the mixture for 30 - 40 min, and then heat in a polytetrafluoroethylene-lined autoclave at 90 - 100 °C for 14 - 18 h; then, wash the filtered solid composite material with pure water 3 - 5 times, wash with ethanol 1 - 2 times, and dry under vacuum conditions at 50 - 60 °C to obtain the resin-MOFs product; (2) The product of the first step was combined with 20 - 30 mL of a 1 - 2 mg / mL nitrile hydratase solution in a buffer solution of 80 - 120 mM phosphate buffer (pH = 7.0). The enzyme was immobilized by electrostatic interaction, washed 3 - 5 times with pure water, and then a resin - MOFs - enzyme system was obtained after vacuum freeze - drying. The final product was placed in 80 - 120 mM phosphate buffer (pH = 7.0) and stored at 25 - 35 °C to determine its thermal stability; (3) At 10 - 20 °C, 45 - 55 g of an acrylonitrile solution with a concentration of 1000 - 1500 mmol / L prepared with 80 - 120 mM phosphate buffer at pH = 7 was added to a 200 mL conical flask. 0.1 - 2.5 g of the resin - MOFs - enzyme system obtained in step (2) was added, and the reaction was carried out under magnetic stirring at 200 - 400 r / min for 50 - 70 min. Then, 0.6 - 1.0 mL of 6 mol / L HCl was added to terminate the reaction, and then the acrylamide concentration was measured; The resin is one of D201 and D301 resins, the metal salt is one of nickel nitrate hexahydrate, cobalt nitrate, and ferric chloride hexahydrate, the ligand is one of imidazole, 2 - methylimidazole, and terephthalic acid, and the solvent is one of N,N - dimethylformamide (DMF), methanol, and pure water.

[0006] Compared with the prior art, the present invention has the following benefits: (1) The resin - MOFs - enzyme system has high stability and low substrate - product diffusion resistance. The nitrile hydratase is adsorbed in the pores and on the surface of the resin loaded with MOF through electrostatic and adsorption interactions, and has the ability to efficiently catalyze the conversion of acrylonitrile to acrylamide.

[0007] (2) The resin - MOFs - enzyme system has high porosity, specific surface area, and high loading capacity. Immobilizing the nitrile hydratase on the surface of the resin loaded with MOF will significantly improve the stability of the system, making the conversion of acrylonitrile to acrylamide more efficient.

[0008] (3) The resin - MOFs - enzyme system has good biocompatibility and high thermal stability. Therefore, it has a higher half - life at high temperatures. Description of the Drawings

[0009] Figure 1 It is a mechanism diagram of the conversion of acrylonitrile to acrylamide by the resin - MOFs - enzyme system; Figure 2 It is a graph of the enzyme - time activity change in the product of Example 1; Figure 3 It is a graph of the enzyme - time activity change in the product of Example 2; Figure 4 Graph of enzyme - time activity change in the product of Example 3; Figure 5 Graph of enzyme - time activity change in the product of Example 4; Figure 6 Graph of enzyme - time activity change in the product of Example 5; Figure 7 Graph of enzyme - time activity change in the product of Example 6; Figure 8 Graph of enzyme - time activity change in the product of Comparative Example 1; Figure 9 Graph of enzyme - time activity change in the product of Comparative Example 2; Figure 10 Graph of the change trend of acrylamide content over time at different acrylonitrile concentrations in Example 1; Figure 11 Graph of the change trend of acrylamide content over time at different acrylonitrile concentrations in Example 2; Figure 12 Graph of the change trend of acrylamide content over time at different acrylonitrile concentrations in Example 3; Figure 13 Graph of the change trend of acrylamide content over time at different acrylonitrile concentrations in Example 4; Figure 14 Graph of the change trend of acrylamide content over time at different acrylonitrile concentrations in Example 5; Figure 15 Graph of the change trend of acrylamide content over time at different acrylonitrile concentrations in Example 6; Figure 16 Graph of the change trend of acrylamide content over time at different acrylonitrile concentrations in Comparative Example 1; Figure 17 Graph of the change trend of acrylamide content over time at different acrylonitrile concentrations in Comparative Example 2. Detailed implementation manners

[0010] The following are specific examples of the present invention, further describing the operation scheme of the present invention. However, the protection scope of the present invention includes but is not limited to these examples. All other examples obtained by those of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

[0011] Example 1: (1) Transfer 2.1 parts of D201 resin, 0.32 parts of terephthalic acid, and 0.2 parts of sodium hydroxide to pure water in sequence. After slowly stirring for 1 h, wash it 3 times with pure water until neutral. Dissolve 0.52 parts of ferric chloride hexahydrate in 30 parts of N,N-dimethylformamide (DMF), and then transfer the above-washed resin into the metal salt solution. Stir the mixture for 35 min, and then heat it in a Teflon-lined autoclave at 100 °C for 14 h. Then, wash the filtered solid composite material 3 times with pure water and 2 times with ethanol, and dry it under vacuum at 50 °C to obtain the resin-MOF product; (2) Combine the product of the first step with 22 mL of 1.6 mg / mL nitrile hydratase solution in 100 mM phosphate buffer (pH = 7.0), immobilize the enzyme by electrostatic interaction, wash it 3 times with pure water, and obtain the resin-MOFs-enzyme system after vacuum freeze-drying. Place the final product in 100 mM phosphate buffer (pH = 7.0), store it at 35 °C, and measure its thermal stability; (3) At 15 °C, add 50 g of acrylonitrile solutions with concentrations of 1000, 1250, and 1500 mmol / L prepared with 100 mM pH = 7 phosphate buffer into a 200 mL conical flask respectively. Add 2.1 g of the resin-MOFs-enzyme system obtained in step (2) to each portion. Under magnetic stirring at 300 r / min, react for 60 min, then add 0.8 mL of 6 mol / L HCl to terminate the reaction, and finally measure the acrylamide concentrations of the three portions.

[0012] Example 2: (1) Transfer 2 parts of D201 resin and 0.4 parts of 2-methylimidazole to pure water in sequence, and stir slowly for 1 h. Dissolve 0.55 parts of cobalt nitrate in 30 parts of methanol, and then transfer the above-washed resin into the metal salt solution. Stir the mixture for 35 min, and then heat it in a Teflon-lined autoclave at 100 °C for 14 h. Then, wash the filtered solid composite material 3 times with pure water and 2 times with ethanol, and dry it under vacuum at 50 °C to obtain the resin-MOF product; (2) Combine the product of the first step with 28 mL of 1.4 mg / mL nitrile hydratase solution in 100 mM phosphate buffer with pH 7.0, immobilize the enzyme by electrostatic interaction, wash it 3 times with pure water, and obtain the resin-MOFs-enzyme system after vacuum freeze-drying. Place the final product in 100 mM phosphate buffer (pH = 7.0), store it at 35 °C, and measure its thermal stability; (3) At 15 °C, add 50 g of three acrylonitrile solutions with concentrations of 1000, 1250, and 1500 mmol / L respectively, which are prepared with 100 mM phosphate buffer solution with pH = 7, to a 200 mL conical flask. Add 2 g of the resin-MOFs-enzyme system from step (2) to each portion. Under magnetic stirring at 300 r / min, react for 60 min, then add 0.8 mL of 6 mol / L HCl to terminate the reaction, and finally measure the acrylamide concentrations of the three portions.

[0013] Example 3: (1) Transfer 2.3 portions of D201 resin and 0.38 portion of imidazole to pure water in sequence, and stir slowly for 1 h. Dissolve 0.53 portion of nickel nitrate hexahydrate in 30 portions of pure water, then transfer the washed resin above to the metal salt solution, stir the mixture for 35 min, and then heat in a Teflon-lined autoclave at 100 °C for 14 h. Then, wash the filtered solid composite material 3 times with pure water, 2 times with ethanol, and dry it at 50 °C under vacuum conditions to obtain the resin-MOF product; (2) Compound the product of the first step with 25 mL of 1.5 mg / mL nitrile hydratase solution in 100 mM phosphate buffer solution with pH = 7.0, immobilize the enzyme by electrostatic interaction, wash it 3 times with pure water, and obtain the resin-MOFs-enzyme system after vacuum freeze-drying. Place the final product in 100 mM phosphate buffer solution (pH = 7.0), store it at 35 °C, and measure its thermal stability; (3) At 15 °C, add 50 g of three acrylonitrile solutions with concentrations of 1000, 1250, and 1500 mmol / L respectively, which are prepared with 100 mM phosphate buffer solution with pH = 7, to a 200 mL conical flask. Add 2.2 g of the resin-MOFs-enzyme system from step (2) to each portion. Under magnetic stirring at 300 r / min, react for 60 min, then add 0.8 mL of 6 mol / L HCl to terminate the reaction, and finally measure the acrylamide concentrations of the three portions.

[0014] Example 4: (1) Transfer 2.4 portions of D301 resin, 0.5 portion of terephthalic acid, and 0.2 portion of sodium hydroxide to pure water in sequence. After stirring slowly for 1 h, wash it 3 times with pure water until neutral. Dissolve 0.6 portion of ferric chloride hexahydrate in 30 portions of DMF, then transfer the washed resin above to the metal salt solution, stir the mixture for 35 min, and then heat in a Teflon-lined autoclave at 100 °C for 16 h. Then, wash the filtered solid composite material 3 times with pure water, 2 times with ethanol, and dry it at 50 °C under vacuum conditions to obtain the resin-MOF product; (2) The product of the first step was combined with 20 mL of a 2 mg / mL nitrile hydratase solution in a 100 mM phosphate buffer solution with a pH of 7.0. The enzyme was immobilized by electrostatic interaction, washed 3 times with pure water, and then freeze-dried under vacuum to obtain a resin-MOFs-enzyme system. The final product was placed in 100 mM phosphate buffer (pH = 7.0) and stored at 35 °C to determine its thermal stability. (3) At 15 °C, 50 g of acrylonitrile solutions with concentrations of 1000, 1250, and 1500 mmol / L respectively, prepared with 100 mM phosphate buffer (pH = 7), were added to a 200 mL conical flask. 2 g of the resin-MOFs-enzyme system obtained in step (2) was added to each portion. The reaction was carried out for 60 min under magnetic stirring at 300 r / min, then 0.8 mL of 6 mol / L HCl was added to terminate the reaction, and finally the concentrations of acrylamide in the three portions were measured.

[0015] Example 5: (1) 2.2 parts of D301 resin and 0.38 part of 2-methylimidazole were successively transferred into pure water and stirred slowly for 1 h. 0.62 part of cobalt nitrate was dissolved in 30 parts of methanol, and then the washed resin was transferred into the metal salt solution. The mixture was stirred for 35 min and then heated in a Teflon-lined autoclave at 100 °C for 16 h. Then, the filtered solid composite material was washed 3 times with pure water, 2 times with ethanol, and dried under vacuum at 50 °C to obtain a resin-MOF product. (2) The product of the first step was combined with 24 mL of a 1.6 mg / mL nitrile hydratase solution in a 100 mM phosphate buffer solution with a pH of 7.0. The enzyme was immobilized by electrostatic interaction, washed 3 times with pure water, and then freeze-dried under vacuum to obtain a resin-MOFs-enzyme system. The final product was placed in 100 mM phosphate buffer (pH = 7.0) and stored at 35 °C to determine its thermal stability. (3) At 15 °C, 50 g of acrylonitrile solutions with concentrations of 1000, 1250, and 1500 mmol / L respectively, prepared with 100 mM phosphate buffer (pH = 7), were added to a 200 mL conical flask. 2.3 g of the resin-MOFs-enzyme system obtained in step (2) was added to each portion. The reaction was carried out for 60 min under magnetic stirring at 300 r / min, then 0.8 mL of 6 mol / L HCl was added to terminate the reaction, and finally the concentrations of acrylamide in the three portions were measured.

[0016] Example 6: (1) Transfer 2 parts of D301 resin and 0.45 part of imidazole to pure water in sequence, and stir slowly for 1 h. Dissolve 0.68 part of nickel nitrate hexahydrate in 30 parts of pure water, then transfer the above-washed resin to the metal salt solution, stir the mixture for 35 min, and then heat it in a PTFE-lined autoclave at 100 °C for 16 h. Then, wash the filtered solid composite material 3 times with pure water, 2 times with ethanol, and dry it under vacuum at 50 °C to obtain the resin-MOF product; (2) Combine the product of the first step with 30 mL of 1.2 mg / mL nitrile hydratase solution in 100 mM phosphate buffer solution with pH = 7.0, immobilize the enzyme by electrostatic interaction, wash it 3 times with pure water, and obtain the resin-MOFs-enzyme system after vacuum freeze-drying. Place the final product in 100 mM phosphate buffer (pH = 7.0), store it at 35 °C, and measure its thermal stability; (3) At 15 °C, add 50 g of acrylonitrile solutions with concentrations of 1000, 1250, and 1500 mmol / L, respectively, prepared with 100 mM phosphate buffer with pH = 7 in a 200 mL conical flask, add 2.1 g of the resin-MOFs-enzyme system from step (2), and react under magnetic stirring at 300 r / min for 60 min. Then add 0.8 mL of 6 mol / L HCl to terminate the reaction, and finally measure the concentrations of the three acrylamide samples.

[0017] Comparative Example 1: (1) Transfer 0.36 part of terephthalic acid and 0.22 part of sodium hydroxide to pure water in sequence. Stir slowly for 1 h until neutral. Dissolve 0.65 part of ferric chloride hexahydrate in 30 parts of DMF, then transfer the above ligand solution to the metal salt solution, stir the mixture for 30 min, and then heat it in a PTFE-lined autoclave at 60 °C for 3 h. Then, wash the filtered solid composite material three times with pure water, once with ethanol, and dry it under vacuum at 60 °C to obtain the MOF product; (2) Combine the product of the first step with 28 mL of 1.3 mg / mL nitrile hydratase solution in 50 mL of 100 mM phosphate buffer solution with pH = 7.0, wash it 3 times with pure water, and obtain the final product after vacuum freeze-drying. Place the final product in 100 mM phosphate buffer (pH = 7.0), store it at 35 °C, and measure its thermal stability; (3) At 15 °C, add 50 g of three acrylonitrile solutions with concentrations of 1000, 1250, and 1500 mmol / L respectively, which are prepared with 100 mM phosphate buffer solution with pH = 7, to a 200 mL conical flask. Add 1.8 g of the resin-MOFs-enzyme system from step (2). Under magnetic stirring at 300 r / min, react for 60 min, then add 0.8 mL of 6 mol / L HCl to terminate the reaction, and finally measure the acrylamide concentrations of the three portions.

[0018] Comparative Example 2: (1) Combine 25 mL of 1.5 mg / mL nitrile hydratase solution with 2 portions of D201 resin in a phosphate buffer solution with pH = 7.0, wash it 3 times with pure water, and obtain the product after vacuum freeze-drying. (2) Place the final product in 100 mM phosphate buffer (pH = 7.0), store it at 35 °C, and measure its thermal stability. (3) At 15 °C, add 50 g of three acrylonitrile solutions with concentrations of 1000, 1250, and 1500 mmol / L respectively, which are prepared with 100 mM phosphate buffer solution with pH = 7, to a 200 mL conical flask. Add 2.5 g of the resin-MOFs-enzyme system from step (2) to each portion. Under magnetic stirring at 300 r / min, react for 60 min, then add 0.8 mL of 6 mol / L HCl to terminate the reaction, and finally measure the acrylamide concentrations of the three portions.

[0019] The thermal stability results of the above Examples 1-6 and Comparative Examples 1-2 are shown in the following figure attached Figure 2-9 As shown, the results indicate that since nitrile hydratase is uniformly adsorbed inside and on the surface of the resin pores through electrostatic interactions, and the protection of the enzyme is further enhanced by the presence of MOF. Therefore, the resin-MOFs-enzyme systems of Examples 1-6 have good thermal stability, with a half-life of approximately 80 - 90 h at 35 °C, thus having good thermal stability. However, the products of the comparative examples have poor thermal stability and are not conducive to long-term use.

[0020] The catalytic effects of the above Examples 1-6 and Comparative Examples 1-2 are shown in the following figure attached Figure 10-17As shown, the results indicate that since the nitrile hydratase is evenly distributed in the pores and on the surface of the resin, and the specific surface area is further increased by relying on the MOF, the catalytic efficiency of the enzyme for acrylonitrile is effectively increased. Therefore, at pH = 7 and 15 °C, Examples 1-6 all show relatively high conversion efficiencies. Among them, when the acrylonitrile concentration is 1250 mmol / L, the conversion efficiency is the best and the final acrylamide concentration is the highest, approximately 940 mmol / L. In Comparative Example 1, since the enzyme and the MOF are not loaded on the resin, the relative contact area with the acrylonitrile solution is reduced. In Comparative Example 2, since there is no MOF-assisted immobilization and loading of the enzyme, the enzyme content on the resin is low. Therefore, the conversion efficiencies of Comparative Examples 1-2 for acrylonitrile are relatively low, and the final acrylamide concentration is around 400 mmol / L.

Claims

1. A method for preparing acrylamide by biological method by constructing a resin-MOFs-enzyme system, characterized in that: The preparation method thereof comprises the following steps: (1) 1.5-2.5 parts of resin, 0.2-0.6 parts of ligand and 0-0.5 parts of sodium hydroxide are transferred to pure water in sequence by mass, slowly stirred for 1-2 hours, and then washed with pure water for 3-5 times until neutral; 0.3-0.8 parts of metal salt are dissolved in 25-35 parts of solvent, and then the washed resin is transferred to the metal salt solution, the mixture is stirred for 30-40 minutes, and then heated in a polytetrafluoroethylene-lined autoclave at 90-100 °C for 14-18 hours; then, the filtered solid composite material is washed with pure water for 3-5 times, washed with ethanol for 1-2 times, and dried at 50-60 °C under vacuum conditions to obtain a resin-MOFs product; (2) Compounding the product of step (1) with 20-30 mL of 1-2 mg / mL nitrile hydratase solution in 80-120 mM phosphate buffer at pH = 7, immobilizing the enzyme by electrostatic action, washing it with pure water 3-5 times, and obtaining a resin-MOFs-enzyme system after vacuum freeze drying. The final product is placed in 80-120 mM phosphate buffer (pH = 7.0), stored at 25-35 ° C, and its thermal stability is determined; (3) At 10-20 °C, add 45-55 g of acrylonitrile solution with a concentration of 1000-1500 mmol / L prepared in 80-120 mM pH=7 phosphate buffer to a 200 mL conical flask, then add 0.1-2.5 g of the resin-MOFs-enzyme system of step (2), react under magnetic stirring at 200-400 r / min for 50-70 min, then add 0.6-1.0 mL 6 mol / L HCl to terminate the reaction, and then measure the acrylamide concentration.

2. The method for preparing acrylamide by biological method using a resin-MOFs-enzyme system according to claim 1, characterized in that: The resin is one of D201 and D301 resins, the metal salt is one of nickel nitrate hexahydrate, cobalt nitrate and ferric chloride hexahydrate, and the ligand is one of imidazole, 2-methylimidazole and terephthalic acid.

3. The method for preparing acrylamide by biological method using a resin-MOFs-enzyme system according to claim 1, characterized in that: The solvent is one of N,N-dimethylformamide (DMF), methanol and pure water.

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