Catalytic application process of nitrile hydratase for efficiently preparing acrylamide

Abstract

The invention relates to a nitrile hydratase catalysis application process for efficiently preparing acrylamide, and relates to the technical field of acrylamide preparation, and the nitrile hydratase catalysis application process comprises the following steps: adding a nitrile hydratase strain into a seed culture medium for culturing to obtain a primary culture solution; adding the primary culture solution into a fermentation culture medium for fermentation to obtain a fermentation solution; preparing a nitrile hydratase catalyst; the method comprises the following steps: adding acrylonitrile into water, adding a nitrile hydratase catalyst to obtain a reaction solution, and carrying out hydration reaction to obtain a product mixed solution; removing the nitrile hydratase catalyst from the product mixed solution through magnetic separation to obtain an acrylamide hydrated solution; and filtering the acrylamide hydration liquid through an ultrafiltration membrane to obtain an acrylamide crude product liquid, refining the crude product liquid, and concentrating to obtain an acrylamide finished product. The method can be used for efficiently preparing acrylamide, and has the effect of improving the yield of acrylamide.

Description

Technical Field

[0001] The present application relates to the technical field of acrylamide preparation, and in particular to a catalytic application process of nitrile hydratase for efficiently preparing acrylamide. Background Art

[0002] As the core monomer of high-molecular materials such as polyacrylamide, the traditional preparation process of acrylamide has long relied on chemical catalysis. Taking the sulfuric acid hydration method as an example, this process generates acrylamide sulfate through the hydrolysis reaction of acrylonitrile and sulfuric acid, and then obtains the target product through ammonia neutralization. However, this process not only produces a large amount of low-value ammonium sulfate as a by-product, but also faces serious problems of equipment corrosion and treatment of three wastes. Although the catalytic hydration method uses a copper-based catalyst to achieve the reaction under milder conditions, high-temperature and high-pressure operations still result in high energy consumption, and it is difficult to regenerate the catalyst, and additional purification steps are required for the metal ions remaining in the product. The common pain points of these traditional processes are that it is difficult to balance efficiency, economy and environmental friendliness. Especially in the context of increasingly strict environmental protection regulations, the development of green synthesis technologies has become an urgent need in the industry.

[0003] The introduction of biocatalysis technology brings an innovation opportunity for acrylamide preparation. As a highly selective biocatalyst, nitrile hydratase can catalyze the direct hydration of acrylonitrile to acrylamide under normal temperature and pressure, with advantages such as high conversion rate, high product purity and no need for heavy metals. For example, immobilized Rhodococcus nitrile hydratase can control the acrylonitrile concentration at a low level under mild conditions, realize the continuous production of acrylamide solution, and the content of by-products is extremely low. However, the existing biocatalytic process still has the problem of low enzyme catalytic efficiency, which in turn affects the yield and restricts industrial application, so it needs to be improved. Summary of the Invention

[0004] In order to improve the preparation efficiency of acrylamide, the present application provides a catalytic application process of nitrile hydratase for efficiently preparing acrylamide.

[0005] A catalytic application process of nitrile hydratase for efficiently preparing acrylamide provided by the present application adopts the following technical scheme: A catalytic application process of nitrile hydratase for efficiently preparing acrylamide, comprising the following steps: (1) Strain culture: adding nitrile hydratase strain to a seed culture medium for culture to obtain a preliminary culture solution; adding the preliminary culture solution to a fermentation culture medium for fermentation to obtain a fermentation broth; (2) Immobilization of the strain: Mix the chitosan solution and the sodium alginate solution, add magnetic nanospheres, adjust the pH to neutral after ultrasonic treatment to obtain a carrier dispersion; after mixing the fermentation broth with the carrier dispersion, drop it into a calcium chloride solution, separate and wash to obtain preliminarily cross-linked microspheres, add the preliminarily cross-linked microspheres to a citric acid solution, separate and wash after reaction to obtain microspheres; add the microspheres to a polyethyleneimine solution, separate and wash after separation to obtain a nitrile hydratase catalyst; (3) Catalytic hydration: Add acrylonitrile to water, add a nitrile hydratase catalyst to obtain a reaction solution, and carry out a hydration reaction to obtain a product mixture; (4) Post-treatment: Remove the nitrile hydratase catalyst from the product mixture by magnetic separation to obtain an acrylamide hydration solution; filter the acrylamide hydration solution through an ultrafiltration membrane to obtain a crude acrylamide solution, refine and concentrate the crude solution to obtain a finished acrylamide product.

[0006] The strain cultivation is based on the principle of microbial metabolic regulation. Through the segmented cultivation of the seed medium and the fermentation medium, a gradient environment from proliferation to enzyme production is provided for the nitrile hydratase strain, promoting the accumulation of cell biomass and the expression of enzyme activity, and providing a high-concentration enzyme source for subsequent catalysis; the strain immobilization uses a chitosan-sodium alginate composite carrier to encapsulate magnetic nanospheres, utilizes the ion cross-linking effect of sodium alginate and calcium chloride to form a gel network, combines the amino groups of chitosan to enhance the hydrophilicity of the carrier, and then adjusts the cross-linking density through a citric acid solution and modifies with a polyethyleneimine solution to introduce positive charges, realizing the dual immobilization of physical embedding and chemical adsorption of enzyme molecules. At the same time, the magnetic microspheres endow the carrier with magnetic response characteristics, facilitating subsequent separation and recovery; the catalytic hydration utilizes the specific catalytic function of nitrile hydratase on acrylonitrile. Through the uniform dispersion of the substrate and the catalyst in the reaction solution, the active center of the enzyme is fully contacted with acrylonitrile molecules, and the hydration reaction is efficiently catalyzed under suitable reaction conditions to generate acrylamide; the post-treatment relies on the superparamagnetic properties of magnetic nanospheres, quickly realizes the solid-liquid separation of the catalyst and the product mixture through magnetic separation, further removes small molecule impurities through ultrafiltration membrane filtration, and then improves the product purity through the refining and concentration process, ensuring the quality of the finished acrylamide product and the cyclic sustainability of the process; the above process improves the efficiency of enzyme-catalyzed preparation of acrylamide and the yield of acrylamide through the synergistic effect of microbial cultivation, carrier design, catalytic reaction and separation technology.

[0007] Preferably, in the step (1), the nitrile hydratase strain is cultured in the seed medium at a temperature of 28 - 32 °C for 8 - 12 h, and then cultured at 24 - 26 °C for 4 - 8 h.

[0008] In the early stage of seed culture, culturing at a relatively high temperature can provide a suitable metabolic environment for microorganisms, accelerate the rate of various biochemical reactions in cells, enable the nitrile hydratase strain to quickly enter the logarithmic growth phase, multiply in large numbers, thereby increasing the number of bacterial cells. An adequate number of bacterial cells is the basis for the subsequent large-scale synthesis of nitrile hydratase. In the later stage of culture, since the process of enzyme synthesis by microorganisms is regulated by various factors, lowering the temperature may activate the expression of certain specific genes, prompting the bacterial cells to use more energy and substances for the synthesis of nitrile hydratase, while avoiding the damage to the enzyme structure that may be caused by high temperature, ensuring the activity of the synthesized enzyme, so that more highly active nitrile hydratase can efficiently catalyze the conversion of acrylonitrile into acrylamide, and improve the yield of acrylamide.

[0009] Preferably, after the preliminary culture solution in step (1) is added to the fermentation medium and cultured for 24 h, pure oxygen is introduced for 20 - 40 s every 1.5 - 2.5 h.

[0010] By optimizing the dissolved oxygen supply in the fermentation process through a periodic pure oxygen introduction strategy, in the later stage of fermentation culture, the bacterial cells enter the peak enzyme production period, and the demand for oxygen increases significantly. At this time, periodic introduction of pure oxygen can break through the dissolved oxygen limitation of traditional continuous ventilation, quickly increase the dissolved oxygen content in the system through short-time high-concentration oxygen pulses, meet the oxygen demand for nitrile hydratase synthesis, and at the same time avoid the oxidative stress of bacterial cells or damage to enzyme active groups caused by long-term high dissolved oxygen; through intermittent enhanced oxygen transfer, it not only ensures the oxidative environment required for enzyme synthesis, but also reduces the interference of continuous ventilation on the metabolic balance of bacterial cells, thereby increasing the yield and activity of nitrile hydratase, providing a highly efficient enzyme source for the subsequent catalytic reaction, and finally achieving an increase in the acrylamide yield by enhancing the catalytic efficiency.

[0011] Preferably, in the carrier dispersion liquid in step (2), the mass ratio of chitosan, sodium alginate and magnetic nanospheres is 2:1:(0.6 - 0.8).

[0012] Chitosan and sodium alginate cooperate with each other to form a suitable network structure. Chitosan has certain rigidity and adsorption properties, while sodium alginate has good film-forming and gelation characteristics. The combination of the two can construct a stable carrier framework, providing a reliable support structure for the immobilization of nitrile hydratase, preventing the loss of the enzyme during the reaction process, and ensuring the activity and stability of the enzyme. The addition of magnetic nanospheres endows the carrier with magnetic response characteristics. At the above ratio, the magnetic nanospheres are evenly distributed in the carrier, neither destroying the original structure of the carrier due to excessive amount nor failing to achieve effective magnetic separation due to insufficient amount. In the subsequent post-treatment process, the carrier immobilized with nitrile hydratase can be quickly and efficiently separated from the reaction system by using magnetism, facilitating the recovery and reuse of the catalyst and reducing the production cost. The above mass ratio can adjust the surface properties and pore structure of the carrier. A reasonable surface property is conducive to the binding of the enzyme to the carrier, promoting the contact between the substrate and the active center of the enzyme, and improving the efficiency of the catalytic reaction. A suitable pore structure can provide channels for the diffusion of the substrate and product, reduce the mass transfer resistance, and further enhance the rate of the catalytic reaction and the yield of acrylamide.

[0013] Preferably, the raw materials for preparing the magnetic nanospheres include magnetite, γ-iron oxide, tetraethyl orthosilicate, polyvinyl alcohol, and dopamine.

[0014] As the core magnetic nuclei, magnetite and γ - ferric oxide endow the microspheres with superparamagnetism, ensuring their rapid response and precise recovery in magnetic separation. The combination of the two can better adjust the saturation magnetization intensity and coercivity of the magnetic nanospheres, enabling them to exhibit more suitable magnetic response performance in different magnetic field environments and achieving more efficient catalyst recovery during magnetic separation. Meanwhile, γ - ferric oxide has relatively stable chemical properties. After being compounded with magnetite, it can enhance the overall corrosion resistance of the magnetic nanospheres to a certain extent, reduce the dissolution of metal ions caused by changes in the chemical environment in the reaction system, and thus reduce the interference with the activity of nitrile hydratase. Tetraethyl orthosilicate forms a silica coating layer through hydrolysis and condensation, isolating the magnetic nuclei from the external environment, preventing metal ion leakage from interfering with enzyme activity, and at the same time providing modifiable silanol groups. Polyvinyl alcohol, as a dispersant and stabilizer, inhibits the aggregation of microspheres through steric hindrance effects, ensuring particle size uniformity and enhancing the dispersibility of the carrier in the reaction solution. Dopamine forms a polydopamine coating on the surface of the microspheres by virtue of the strong adhesion of its catechol groups, providing abundant active sites such as amino and phenolic hydroxyl groups. These active sites can bind to the functional groups in chitosan and sodium alginate through covalent bonds or hydrogen bonds, enhancing the binding strength between the microspheres and the composite carrier, and at the same time providing multiple binding sites for nitrile hydratase, improving the enzyme immobilization efficiency and stability. Through the multi - layer structure design and the cooperation of functional groups, the magnetic nanospheres construct a composite carrier with strong magnetic responsiveness, chemical stability and high enzyme - loading capacity, reducing enzyme loss and enhancing the catalytic cycle efficiency, and ultimately achieving the improvement of acrylamide yield and process sustainability.

[0015] Preferably, the magnetic nanospheres are prepared by the following steps: Mix magnetite and γ - ferric oxide, and ultrasonically disperse them in an aqueous solution of polyvinyl alcohol to obtain a dispersion. Add tetraethyl orthosilicate to the dispersion, heat and react, then cool down, add dopamine, adjust the pH to alkaline for reaction, and then perform magnetic separation and washing to obtain the magnetic nanospheres.

[0016] The magnetic nanospheres prepared according to the above steps can effectively improve the production efficiency and yield of acrylamide.

[0017] Preferably, in step (2), cross - link at 3 - 5°C for 12 - 18 min in a calcium chloride solution, cross - link at 28 - 32°C for 15 - 25 min in a citric acid solution, and cross - link at 3 - 5°C for 1.5 - 2.5 h in a polyethylenimine solution.

[0018] Low-temperature crosslinking in calcium chloride solution can enable sodium alginate and calcium ions to slowly form a uniform and stable gel network structure, avoiding the formation of irregular and uneven structures due to too fast crosslinking speed at high temperature, providing a good basic framework for subsequent enzyme immobilization and reducing damage to enzyme activity; high-temperature crosslinking in citric acid solution can accelerate the reaction rate, promote further chemical reactions of the initially crosslinked microspheres, adjust the pore size and surface properties of the microspheres, optimize the contact and diffusion environment between the substrate and the enzyme, and improve the catalytic reaction efficiency; low-temperature crosslinking in polyethyleneimine solution helps the polyethyleneimine molecules to fully bind to the surface of the microspheres, enhance the positive charge density and chemical stability of the microsphere surface, improve the binding force between the enzyme and the carrier, prevent the enzyme from falling off during the catalytic reaction process, thus ensuring the activity and stability of the catalyst and improving the preparation efficiency and yield of acrylamide.

[0019] Preferably, in step (3), the initial concentration of acrylonitrile is 3-5%, the addition amount of nitrile hydratase catalyst is 10-15 g / L, the initial temperature of the reaction solution is 16-20 °C, stirring reaction is carried out at a speed of 200-300 rpm for 0.5-1 h, the temperature is raised to 30-32 °C and the reaction is carried out for 2-3 h. After the acrylamide concentration reaches 15%, acrylonitrile is replenished to maintain the acrylonitrile concentration at 4-6%. After the propionamide concentration reaches 30-40%, the temperature is lowered to 24-26 °C and the reaction is carried out for 2-3 h. During the hydration reaction, the pH is controlled at 7.2-7.8.

[0020] A reasonable initial concentration of acrylonitrile provides a suitable substrate environment for the reaction, which can not only avoid inhibition of the enzyme due to too high concentration, but also prevent too slow reaction rate caused by too low concentration; an appropriate addition amount of nitrile hydratase catalyst ensures sufficient active sites to fully contact the substrate and accelerates the reaction process; the initial low-temperature reaction environment helps to protect the active structure of the enzyme and avoid premature inactivation of the enzyme at high temperature. At the same time, with a certain speed of stirring, the substrate and the catalyst are evenly mixed to enhance the mass transfer effect. Subsequently, raising the temperature can accelerate the reaction rate, promote the rapid progress of the reaction, and increase the amount of acrylamide generated per unit time; when the acrylamide reaches a certain concentration, acrylonitrile is replenished and maintained at an appropriate concentration to continuously provide the substrate for the reaction and ensure that the reaction proceeds in the direction of generating the product; when the acrylamide concentration further increases, lowering the temperature for the reaction can reduce the occurrence of side reactions and improve the selectivity and purity of the product; during the whole hydration reaction, controlling the pH within a suitable range can maintain the stability of the active center structure of the enzyme, make the enzyme in the best catalytic state, thus ensuring the efficient and stable progress of the reaction and finally realizing the efficient preparation of acrylamide.

[0021] Preferably, hydroquinone and copper sulfate are added to the reaction solution in step (3).

[0022] Hydroquinone is used as an inhibitor. Its phenolic hydroxyl group can preferentially combine with acrylonitrile radicals to inhibit the self-polymerization reaction of acrylonitrile at high temperatures, reduce the generation of polyacrylonitrile by-products, and thus improve the selectivity and purity of acrylamide. The copper ions in copper sulfate, as enzyme cofactors, can bind to the amino acid residues at the active center of nitrile hydratase through coordination, optimize the spatial conformation of the enzyme, enhance the affinity and catalytic efficiency of the enzyme for acrylonitrile. At the same time, the redox characteristics of copper ions help to maintain the electron transfer balance in the reaction system and further increase the hydration reaction rate. The synergistic effect of the two not only reduces substrate loss and impurity generation but also enhances the catalytic activity of the enzyme, thereby increasing the yield and quality of acrylamide and reducing the difficulty of subsequent separation and purification.

[0023] Preferably, in the step (4), separation is carried out with a strong magnetic field of 2 - 3 T for 5 - 10 min, and the flow rate of the product mixture is 0.5 - 0.8 m 3 / h, and then washing and separation are carried out with an intensity of 0.5 - 1 T for 10 - 15 min, and the washing flow rate is 0.8 - 1.0 m 3 / h.

[0024] First, separation is carried out using a strong magnetic field intensity. The high magnetic field gradient generates a strong attraction for the magnetic nitrile hydratase catalyst, quickly capturing the carrier microspheres in the reaction solution, and achieving efficient solid-liquid separation of the catalyst and the product mixture in a short time. The high flow rate design ensures the processing efficiency in large-scale production. Subsequently, the magnetic field intensity is switched to a low level for washing and separation. By reducing the magnetic attraction, excessive agglomeration of the catalyst is avoided. At the same time, a relatively high washing flow rate is used to wash away the impurities attached to the surface of the microspheres, achieving fine cleaning of the catalyst under mild conditions. This segmented process not only utilizes the high capture ability of the strong magnetic field to shorten the main separation time but also reduces impurity residues and catalyst damage through weak magnetic washing. Combining flow rate control balances the separation efficiency and washing effect, ultimately improving the recovery purity and activity retention rate of the catalyst, ensuring the cyclic stability of the process and the product quality, and enhancing the preparation efficiency of acrylamide.

[0025] In summary, the present application includes at least one of the following beneficial technical effects: 1. The strain cultivation is based on the principle of microbial metabolic regulation. Through the segmented cultivation of the seed medium and the fermentation medium, a gradient environment from proliferation to enzyme production is provided for the nitrile hydratase strain, promoting the accumulation of cell biomass and the expression of enzyme activity, and providing a high-concentration enzyme source for subsequent catalysis. The strain immobilization uses a chitosan-sodium alginate composite carrier to encapsulate magnetic nanospheres. The ion cross-linking of sodium alginate and calcium chloride is used to form a gel network, and the amino group of chitosan is combined to enhance the hydrophilicity of the carrier. Then, the cross-linking density is adjusted by a citric acid solution, and positive charges are introduced by modification with a polyethyleneimine solution to achieve the dual immobilization of physical entrapment and chemical adsorption of enzyme molecules. At the same time, the magnetic microspheres endow the carrier with magnetic response characteristics, facilitating subsequent separation and recovery. The catalytic hydration utilizes the specific catalytic function of nitrile hydratase for acrylonitrile. Through the uniform dispersion of the substrate and the catalyst in the reaction solution, the active center of the enzyme is fully contacted with acrylonitrile molecules, and the hydration reaction is efficiently catalyzed under suitable reaction conditions to produce acrylamide. The post-treatment relies on the superparamagnetic properties of magnetic nanospheres. Through magnetic separation, the solid-liquid separation of the catalyst and the product mixture is quickly achieved. Ultrafiltration membrane filtration further removes small-molecule impurities, and then the product purity is improved through a refining and concentration process to ensure the quality of the acrylamide finished product and the cyclic sustainability of the process. Through the synergistic effect of microbial cultivation, carrier design, catalytic reaction, and separation technology, the above process improves the efficiency of enzyme-catalyzed preparation of acrylamide and increases the yield of acrylamide.

[0026] 2. Chitosan and sodium alginate cooperate with each other to form a suitable network structure. Chitosan has certain rigidity and adsorption properties, and sodium alginate has good film-forming and gelation characteristics. The combination of the two can construct a stable carrier framework, providing a reliable support structure for immobilizing nitrile hydratase, preventing the loss of the enzyme during the reaction process, and ensuring the activity and stability of the enzyme. The addition of magnetic nanospheres endows the carrier with magnetic response characteristics. At the above ratio, the magnetic nanospheres are evenly distributed in the carrier, neither destroying the original structure of the carrier due to excessive amount nor failing to achieve effective magnetic separation due to insufficient amount. In the subsequent post-treatment process, the carrier immobilized with nitrile hydratase can be quickly and efficiently separated from the reaction system by using magnetism, facilitating the recovery and reuse of the catalyst and reducing the production cost. This specific mass ratio can adjust the surface properties and pore structure of the carrier. A reasonable surface property is conducive to the binding of the enzyme and the carrier, promoting the contact between the substrate and the active center of the enzyme, and improving the efficiency of the catalytic reaction. A suitable pore structure can provide channels for the diffusion of the substrate and the product, reducing the mass transfer resistance, and further enhancing the rate of the catalytic reaction and the yield of acrylamide.

[0027] 3. As an inhibitor, hydroquinone's phenolic hydroxyl group can preferentially combine with acrylonitrile free radicals, inhibiting the self-polymerization reaction of acrylonitrile at high temperatures, reducing the generation of polyacrylonitrile by-products, and thus improving the selectivity and purity of acrylamide; the copper ions in copper sulfate, as enzyme cofactors, can bind to the amino acid residues at the active center of nitrile hydratase through coordination, optimizing the spatial conformation of the enzyme, enhancing the enzyme's affinity for acrylonitrile and catalytic efficiency. At the same time, the redox properties of copper ions contribute to maintaining the electron transfer balance in the reaction system, further increasing the hydration reaction rate; the synergistic effect of the two not only reduces substrate loss and impurity generation but also enhances the catalytic activity of the enzyme, thereby improving the yield and quality of acrylamide and reducing the difficulty of subsequent separation and purification. Detailed implementation mode

[0028] The embodiment of the present application discloses a nitrile hydratase-catalyzed application process for efficiently preparing acrylamide. The raw materials used in this application can be obtained from commercially available raw materials except as otherwise specified. The following further details the present application in combination with examples: Raw material description: Iron tetroxide (CAS No.: 1317-61-9), particle size 30nm, gamma-iron oxide (CAS No.: 1309-37-1), particle size 30nm, polyvinyl alcohol (CAS No.: 9002-89-5), tetraethyl orthosilicate (CAS No.: 78-10-4), dopamine (CAS No.: 51-61-6), citric acid (CAS No.: 77-92-9), polyethylenimine (CAS No.: 9002-98-6), and the nitrile hydratase strain is Nocardia, purchased from Shenzhen Yimi Biotechnology Co., Ltd.

[0029] Example 1 Preparation of magnetic nanospheres Mix 5g of iron tetroxide with 2.5g of gamma-iron oxide, ultrasonically disperse it in an aqueous solution of polyvinyl alcohol with a mass concentration of 3%, and the solid-liquid ratio is 1:15 to obtain a dispersion; add 2g of tetraethyl orthosilicate to the dispersion, react at 60°C for 3h, cool down to 30°C, add 0.6g of dopamine, adjust the pH to 8.5, react for 12h, and after magnetic separation, wash with deionized water to obtain magnetic nanospheres.

[0030] Nitrile hydratase-catalyzed application process for efficiently preparing acrylamide (1) Bacterial culture: the nitrile hydratase bacteria were added to a seed culture medium and cultured at 28° C. for 12 h, then cooled to 24° C. and cultured for 8 h to obtain a preliminary culture solution, wherein the seed culture medium consisted of 20 g / L glucose, 10 g / L peptone, 5 g / L yeast extract, 2 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, 0.01 g / L ferric sulfate heptahydrate, 0.002 g / L manganese chloride tetrahydrate and 0.05 g / L Tween-80; the preliminary culture solution was inoculated into a fermentation medium at a volume content of 5%, and after culturing at 30° C. for 24 h, pure oxygen was introduced for 20 s every 1.5 h, and the pure oxygen flow rate was 5 m / s. 3 / h, and after continuing fermentation for 48h, a fermentation broth was obtained. The fermentation medium consisted of 15g / L glucose, 5g / L soybean cake hydrolyzate, 3g / L yeast extract, 1.5g / L potassium dihydrogen phosphate, 0.5g / L magnesium sulfate heptahydrate, 0.005g / L cobalt chloride hexahydrate, 0.002g / L zinc sulfate heptahydrate, 0.1g / L acrylonitrile and 0.2g / L Tween-80.

[0031] (2) Immobilization of bacterial strains: chitosan acetate solution with a mass concentration of 2% and sodium alginate aqueous solution with a mass concentration of 1% were mixed, magnetic nanoparticles were added, ultrasonicated for 15 minutes, and the pH was adjusted to neutral to obtain a carrier dispersion. The mass ratio of chitosan, sodium alginate and magnetic nanoparticles in the carrier dispersion was 2:1:0.6; the fermentation broth was mixed with the carrier dispersion, and then dripped into a calcium chloride solution, cross-linked at 3°C for 18 minutes, filtered, and washed with deionized water to obtain preliminary cross-linked microspheres, and the preliminary cross-linked microspheres were added to a citric acid solution, cross-linked at 28°C for 25 minutes, filtered, and washed with deionized water to obtain microspheres; the microspheres were added to a polyethyleneimine solution, cross-linked at 3°C for 2.5 hours, and washed with deionized water after magnetic separation to obtain a nitrile hydratase catalyst, which was stored in a 50 mmol / L Tris-HCl buffer (pH 7.5) containing 10% glycerol at 4°C for later use; (3) Catalytic hydration: acrylonitrile was added to water, the initial concentration of acrylonitrile was 3%, and a nitrile hydratase catalyst was added in an amount of 10 g / L to obtain a reaction solution. The initial temperature of the reaction solution was 16° C., and the reaction was stirred at a speed of 200 rpm for 1 hour. The temperature was raised to 30° C. and the reaction was carried out for 3 hours. After the acrylamide concentration reached 15%, acrylonitrile was added to maintain the acrylonitrile concentration at 4%. After the acrylamide concentration reached 30%, the temperature was lowered to 24° C. and the reaction was carried out for 3 hours. During the hydration reaction, the pH was controlled to be 7.2-7.8 to obtain a product mixed solution. (4) Post-treatment: The product mixture was subjected to magnetic separation to remove the nitrile hydratase catalyst to obtain an acrylamide hydration solution, which was separated in a strong magnetic field of 2 T for 10 min. The flow rate of the product mixture was 0.5 m / s. 3 / h, and then wash and separate at a strength of 0.5T for 15min, with a washing flow rate of 1.0m 3 / h, the recovered nitrile hydratase catalyst can be reused, and the enzyme activity retention rate is above 90%; the acrylamide hydration liquid is filtered through an ultrafiltration membrane to obtain a crude acrylamide liquid, the ultrafiltration membrane is made of polyethersulfone, has a molecular weight cutoff of 5kDa, and an operating pressure of 0.4MPa, and the crude liquid is refined by gradient cooling, from 50°C to 30°C at a rate of 0.5°C / min, and then to 4°C at a rate of 1°C / min. After purification, vacuum film is used for evaporation and concentration at 45°C to obtain a finished acrylamide product.

[0032] Example 2 Preparation of magnetic nanoparticles 5 g of ferrosoferric oxide and 2.5 g of γ-ferric oxide were mixed, and ultrasonically dispersed into a polyvinyl alcohol aqueous solution with a mass concentration of 3% and a solid-liquid ratio of 1:15 to obtain a dispersion; 2 g of tetraethyl orthosilicate was added to the dispersion, reacted at 60°C for 3 h, cooled to 30°C, 0.6 g of dopamine was added, the pH was adjusted to 8.5, reacted for 12 h, and washed with deionized water after magnetic separation to obtain magnetic nanoparticles.

[0033] High-efficiency preparation of acrylamide by nitrile hydratase catalysis (1) Bacterial culture: the nitrile hydratase bacteria were added to a seed culture medium and cultured at 32° C. for 8 h, then cooled to 26° C. and cultured for 4 h to obtain a preliminary culture solution, wherein the seed culture medium consisted of 20 g / L glucose, 10 g / L peptone, 5 g / L yeast extract, 2 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, 0.01 g / L ferric sulfate heptahydrate, 0.002 g / L manganese chloride tetrahydrate and 0.05 g / L Tween-80; the preliminary culture solution was inoculated into a fermentation medium at a volume content of 5%, and after culturing at 30° C. for 24 h, pure oxygen was introduced for 40 s every 2.5 h, and the pure oxygen flow rate was 5 m / s. 3 / h, and after continuing fermentation for 48h, a fermentation broth was obtained. The fermentation medium consisted of 15g / L glucose, 5g / L soybean cake hydrolyzate, 3g / L yeast extract, 1.5g / L potassium dihydrogen phosphate, 0.5g / L magnesium sulfate heptahydrate, 0.005g / L cobalt chloride hexahydrate, 0.002g / L zinc sulfate heptahydrate, 0.1g / L acrylonitrile and 0.2g / L Tween-80.

[0034] (2) Immobilization of the strain: Mix a 2% (mass concentration) chitosan acetate solution and a 1% (mass concentration) sodium alginate aqueous solution, add magnetic nanospheres, sonicate for 15 min, adjust the pH to neutral to obtain a carrier dispersion solution. The mass ratio of chitosan, sodium alginate and magnetic nanospheres in the carrier dispersion solution is 2:1:0.8. After mixing the fermentation broth with the carrier dispersion solution, drop it into a calcium chloride solution, carry out a cross-linking reaction at 5 °C for 12 min, filter and wash with deionized water to obtain preliminarily cross-linked microspheres. Add the preliminarily cross-linked microspheres to a citric acid solution, carry out a cross-linking reaction at 32 °C for 15 min, filter and wash with deionized water to obtain microspheres. Add the microspheres to a polyethyleneimine solution, cross-link at 5 °C for 1.5 h, wash with deionized water after magnetic separation to obtain a nitrile hydratase catalyst, and store it in a 50 mmol / L Tris-HCl buffer solution (pH 7.5) containing 10% glycerol at 4 °C for standby; (3) Catalytic hydration: Add acrylonitrile to water, the initial concentration of acrylonitrile is 5%, add a nitrile hydratase catalyst with an addition amount of 15 g / L to obtain a reaction solution. The initial temperature of the reaction solution is 20 °C, stir the reaction at a speed of 300 rpm for 0.5 h, raise the temperature to 32 °C and react for 2 h. After the acrylamide concentration reaches 15%, add acrylonitrile to maintain the acrylonitrile concentration at 6%. After the acrylamide concentration reaches 40%, lower the temperature to 26 °C and react for 2 h. Control the pH at 7.2 - 7.8 during the hydration reaction to obtain a product mixture; (4) Post-treatment: Remove the nitrile hydratase catalyst from the product mixture by magnetic separation to obtain an acrylamide hydration solution, separate it with a strong magnetic field of 3 T for 5 min, the flow rate of the product mixture is 0.8 m 3 / h, then wash and separate with a strength of 1 T for 10 min, and the washing flow rate is 0.8 m 3 / h. The recovered nitrile hydratase catalyst can be reused, and the enzyme activity retention rate is above 90%. Filter the acrylamide hydration solution through an ultrafiltration membrane to obtain a crude acrylamide solution. The ultrafiltration membrane material is polyethersulfone, the cut-off molecular weight is 5 kDa, and the operating pressure is 0.4 MPa. Gradually cool and refine the crude solution, cool from 50 °C to 30 °C at a rate of 0.5 °C / min, and then cool to 4 °C at a rate of 1 °C / min. After refining, concentrate by vacuum thin film evaporation at 45 °C to obtain the finished acrylamide.

[0035] Example 3 Preparation of magnetic nanospheres 5 g of ferrosoferric oxide and 2.5 g of γ-ferric oxide were mixed, and ultrasonically dispersed into a polyvinyl alcohol aqueous solution with a mass concentration of 3% and a solid-liquid ratio of 1:15 to obtain a dispersion; 2 g of tetraethyl orthosilicate was added to the dispersion, reacted at 60°C for 3 h, cooled to 30°C, 0.6 g of dopamine was added, the pH was adjusted to 8.5, reacted for 12 h, and washed with deionized water after magnetic separation to obtain magnetic nanoparticles.

[0036] High-efficiency preparation of acrylamide by nitrile hydratase catalysis (1) Bacterial culture: adding the nitrile hydratase strain to a seed culture medium and culturing it at 30° C. for 10 h, cooling it to 25° C. and culturing it for 6 h to obtain a preliminary culture solution, wherein the seed culture medium is composed of 20 g / L glucose, 10 g / L peptone, 5 g / L yeast extract, 2 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, 0.01 g / L ferric sulfate heptahydrate, 0.002 g / L manganese chloride tetrahydrate and 0.05 g / L Tween-80; the preliminary culture solution is inoculated into a fermentation medium at a volume content of 5%, and after culturing it at 30° C. for 24 h, pure oxygen is introduced for 30 s every 2 h, and the pure oxygen flow rate is 5 m / s. 3 / h, and after continuing fermentation for 48h, a fermentation broth was obtained. The fermentation medium consisted of 15g / L glucose, 5g / L soybean cake hydrolyzate, 3g / L yeast extract, 1.5g / L potassium dihydrogen phosphate, 0.5g / L magnesium sulfate heptahydrate, 0.005g / L cobalt chloride hexahydrate, 0.002g / L zinc sulfate heptahydrate, 0.1g / L acrylonitrile and 0.2g / L Tween-80.

[0037] (2) Immobilization of bacterial strains: chitosan acetate solution with a mass concentration of 2% and sodium alginate aqueous solution with a mass concentration of 1% were mixed, magnetic nanoparticles were added, ultrasonicated for 15 minutes, and the pH was adjusted to neutral to obtain a carrier dispersion. The mass ratio of chitosan, sodium alginate and magnetic nanoparticles in the carrier dispersion was 2:1:0.7; the fermentation broth was mixed with the carrier dispersion, and then dripped into a calcium chloride solution, cross-linked at 4°C for 15 minutes, filtered, and washed with deionized water to obtain preliminary cross-linked microspheres, and the preliminary cross-linked microspheres were added to a citric acid solution, cross-linked at 30°C for 20 minutes, filtered, and washed with deionized water to obtain microspheres; the microspheres were added to a polyethyleneimine solution, cross-linked at 4°C for 2 hours, and washed with deionized water after magnetic separation to obtain a nitrile hydratase catalyst, which was stored in a 50 mmol / L Tris-HCl buffer (pH 7.5) containing 10% glycerol at 4°C for use; (3) Catalytic hydration: Acrylonitrile was added to water with an initial concentration of 4%. A nitrile hydratase catalyst was added at a dosage of 12.5 g / L to obtain a reaction solution. The initial temperature of the reaction solution was 18°C, and it was stirred at a speed of 250 rpm for 0.75 h. Then the temperature was raised to 31°C and reacted for 2.5 h. After the acrylamide concentration reached 15%, acrylonitrile was replenished to maintain the acrylonitrile concentration at 5%. After the acrylamide concentration reached 35%, the temperature was lowered to 25°C and reacted for 2.5 h. During the hydration reaction, the pH was controlled at 7.2 - 7.8 to obtain a product mixture; (4) Post-treatment: The product mixture was passed through magnetic separation to remove the nitrile hydratase catalyst, obtaining an acrylamide hydration solution. It was separated with a strong magnetic field of 2.5 T for 7.5 min, and the flow rate of the product mixture was 0.65 m 3 / h. Then it was washed and separated with an intensity of 0.75 T for 12.5 min, and the washing flow rate was 0.9 m 3 / h. The recovered nitrile hydratase catalyst could be reused, and the enzyme activity retention rate was above 90%. The acrylamide hydration solution was filtered through an ultrafiltration membrane to obtain a crude acrylamide solution. The ultrafiltration membrane was made of polyethersulfone with a cut-off molecular weight of 5 kDa and an operating pressure of 0.4 MPa. The crude solution was refined by gradient cooling, cooling from 50°C to 30°C at a rate of 0.5°C / min, and then to 4°C at a rate of 1°C / min. After refining, it was concentrated by vacuum thin film evaporation at 45°C to obtain the finished acrylamide product.

[0038] Example 4 Example 4 was based on Example 3. The difference between Example 4 and Example 3 was only that in step (1) of Example 4, the nitrile hydratase strain was cultured in the seed medium at 26°C for 14 h, then cooled to 22°C and cultured for 10 h. After the preliminary culture solution was added to the fermentation medium and cultured for 24 h, pure oxygen was introduced for 10 s every 1 h.

[0039] Example 5 Example 5 was based on Example 3. The difference between Example 5 and Example 3 was only that in step (1) of Example 5, the nitrile hydratase strain was cultured in the seed medium at 34°C for 6 h, then cooled to 28°C and cultured for 2 h. After the preliminary culture solution was added to the fermentation medium and cultured for 24 h, pure oxygen was introduced for 50 s every 3 h.

[0040] Example 6 Example 6 was based on Example 3. The difference between Example 6 and Example 3 was only that in step (2) of Example 6, the mass ratio of chitosan, sodium alginate, and magnetic nanospheres in the carrier dispersion was 2:1:0.4.

[0041] Example 7 Example 7 is based on Example 3. The only difference between Example 7 and Example 3 is that in step (2) of Example 7, the mass ratio of chitosan, sodium alginate, and magnetic nanospheres in the carrier dispersion liquid is 2:1:1.

[0042] Example 8 Example 8 is based on Example 3. The only difference between Example 8 and Example 3 is that γ-iron oxide is not added when preparing magnetic nanospheres in Example 8.

[0043] Example 9 Example 9 is based on Example 3. The only difference between Example 9 and Example 3 is that dopamine is not added when preparing magnetic nanospheres in Example 9.

[0044] Example 10 Example 10 is based on Example 3. The only difference between Example 10 and Example 3 is that in step (2) of Example 10, crosslinking is carried out at 0 °C for 20 min in calcium chloride solution, at 25 °C for 30 min in citric acid solution, and at 0 °C for 3 h in polyethyleneimine solution.

[0045] Example 11 Example 11 is based on Example 3. The only difference between Example 11 and Example 3 is that in step (2) of Example 11, crosslinking is carried out at 10 °C for 8 min in calcium chloride solution, at 35 °C for 10 min in citric acid solution, and at 10 °C for 1 h in polyethyleneimine solution.

[0046] Example 12 Example 12 is based on Example 3. The only difference between Example 12 and Example 3 is that in step (3) of Example 12, the initial reaction temperature is 25 °C and the reaction lasts for 2 h.

[0047] Example 13 Example 13 is based on Example 3. The only difference between Example 13 and Example 3 is that 0.05% hydroquinone and 0.005% copper sulfate based on the total mass of the reaction solution are added to the reaction solution in Example 13.

[0048] Comparative Example 1 Comparative Example 1 is based on Example 3. The only difference between Comparative Example 1 and Example 3 is that citric acid is not used for crosslinking in step (2) of Comparative Example 1.

[0049] Comparative Example 2 Comparative Example 2 is based on Example 3. The only difference between Comparative Example 2 and Example 3 is that polyethyleneimine crosslinking is not used in step (2) of Comparative Example 2.

[0050] Performance Detection Test Yield detection of acrylamide: Use a UV spectrophotometer to quantitatively determine the weight of acrylamide in the sample, denoted as A. Calculate the weight of acrylamide that can theoretically be produced from the acrylonitrile raw material used, denoted as B. Calculate the yield = A / B * 100% Table 1 Detection results of acrylamide yield As can be seen from Table 1, the yields of Examples 1 - 3 are greater than 98.2%, indicating that the preparation process of this application can efficiently prepare acrylamide with a high yield.

[0051] As can be seen from Table 1, the only differences between Examples 4 and 5 and Example 3 are that in Examples 4 and 5, the temperature and time of strain cultivation, as well as the frequency and time of oxygen introduction, are regulated. Compared with Example 3, the yields of Examples 4 and 5 have decreased. This is because adjusting the cultivation temperature of the strain will affect the proliferation rate of the bacteria and the enzyme content produced; adjusting the frequency and time of oxygen introduction may affect the enzyme activity, thereby affecting the yield of acrylamide.

[0052] As can be seen from Table 1, the only differences between Examples 6 and 7 and Example 3 are that in Example 6, the mass ratio of chitosan, sodium alginate, and magnetic nanospheres in the carrier dispersion liquid in step (2) is 2:1:0.4, and in Example 7, the mass ratio of chitosan, sodium alginate, and magnetic nanospheres in the carrier dispersion liquid in step (2) is 2:1:1. Compared with Example 3, the performance of Examples 6 and 7 has decreased. This is because adjusting the component ratio in the carrier dispersion liquid, too many or too few magnetic microspheres will affect the magnetic responsiveness of the carrier and the network structure of the catalyst, thereby affecting the catalytic reaction efficiency and thus the yield.

[0053] As can be seen from Table 1, the only differences between Examples 8, 9, 10, and 11 and Example 3 are that in Example 8, γ - iron oxide is not added when preparing magnetic nanospheres, in Example 9, dopamine is not added when preparing magnetic nanospheres, and in Examples 10 and 11, the curing temperature and time are changed during the preparation of the nitrile hydratase catalyst. Compared with Example 3, the yields of Examples 8, 9, 10, and 11 have decreased. This is because without adding γ - iron oxide, the lack of compound synergistic effect reduces the stability of the catalyst; without adding dopamine magnetic nanospheres, the surface lacks active groups, weakening the binding force with sodium alginate and chitosan, and the enzyme immobilization efficiency decreases; changing the curing temperature and time will affect the stability of the nitrile hydratase catalyst, and the mass transfer efficiency and the strength of enzyme immobilization are affected, resulting in a decrease in yield.

[0054] As can be seen from Table 1, the difference between Example 12 and Example 3 is only that: in Example 12, the initial reaction temperature was increased, and compared with Example 3, the performance decreased; this is because increasing the initial reaction temperature will have a certain impact on the enzyme activity due to the exothermic reaction, resulting in a decrease in the yield.

[0055] As can be seen from Table 1, the difference between Example 13 and Example 3 is only that: in Example 13, hydroquinone and copper sulfate were added to the reaction solution, and compared with Example 3, the yield increased; this is because adding hydroquinone and copper sulfate inhibited the self-polymerization of acrylonitrile and further activated the enzyme activity, synergistically enhancing the catalytic conversion of acrylonitrile, resulting in an increase in the yield.

[0056] As can be seen from Table 1, the difference between Comparative Example 1 and Example 3 is only that: in Comparative Example 1, citric acid was not used for cross-linking in step (2), and compared with Example 3, the yield decreased significantly; this is because the lack of citric acid for cross-linking led to a loose carrier network, poor mechanical strength, and a decrease in the enzyme immobilization efficiency, resulting in a significant decrease in the yield.

[0057] As can be seen from Table 1, the difference between Comparative Example 2 and Example 3 is only that: in Comparative Example 2, polyethyleneimine was not used for cross-linking in step (2), and compared with Example 3, the yield decreased significantly; this is because the lack of cross-linking with polyethyleneimine reduced the surface charge of the carrier, weakened the interaction between the substrate and the enzyme during the reaction, resulting in a significant decrease in the yield.

[0058] This specific embodiment is only an explanation of the present application, and it is not a limitation of the present application. Through the above description, relevant staff can make various changes and modifications without departing from the technical idea of this application. The technical scope of this application is not limited to the content in the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A process for the catalytic application of nitrile hydratase for the efficient preparation of acrylamide, characterized in that: It includes the following steps: (1) Strain cultivation: Add the nitrile hydratase strain to the seed medium for cultivation to obtain a preliminary culture solution; add the preliminary culture solution to the fermentation medium for fermentation to obtain a fermentation broth; (2) Strain immobilization: Mix the chitosan solution and the sodium alginate solution, add magnetic nanospheres, adjust the pH to neutral after ultrasonic treatment to obtain a carrier dispersion; mix the fermentation broth with the carrier dispersion, then drop it into the calcium chloride solution, separate and wash to obtain a preliminary cross-linked microsphere, add the preliminary cross-linked microsphere to the citric acid solution, react, separate and wash to obtain a microsphere; add the microsphere to the polyethyleneimine solution, separate and wash to obtain a nitrile hydratase catalyst; (3) Catalytic hydration: Add acrylonitrile to water, add the nitrile hydratase catalyst to obtain a reaction solution, carry out a hydration reaction to obtain a product mixture; (4) Post-treatment: Remove the nitrile hydratase catalyst from the product mixture by magnetic separation to obtain an acrylamide hydration solution; filter the acrylamide hydration solution through an ultrafiltration membrane to obtain a crude acrylamide solution, refine and concentrate the crude solution to obtain a finished acrylamide product.

2. The catalytic application process of nitrile hydratase for efficiently preparing acrylamide according to claim 1, wherein: In the step (1), the nitrile hydratase strain is cultured in the seed medium at a temperature of 28 - 32 °C for 8 - 12 h, and then the temperature is lowered to 24 - 26 °C and cultured for 4 - 8 h.

3. The catalytic application process of nitrile hydratase for efficiently preparing acrylamide according to claim 2, characterized in that: In the step (1), after the preliminary culture solution is added to the fermentation medium and cultured for 24 h, pure oxygen is introduced for 20 - 40 s every 1.5 - 2.5 h.

4. The catalytic application process of nitrile hydratase for efficiently preparing acrylamide according to claim 1, wherein: In the step (2), the mass ratio of chitosan, sodium alginate and magnetic nanospheres in the carrier dispersion is 2:1:(0.6 - 0.8).

5. An application process for the catalytic use of nitrile hydratase for the efficient preparation of acrylamide according to claim 4, characterized in that: The preparation raw materials of the magnetic nanospheres include magnetite, γ - ferric oxide, tetraethyl orthosilicate, polyvinyl alcohol and dopamine.

6. The catalytic application process of nitrile hydratase for efficiently preparing acrylamide according to claim 5, characterized in that: The magnetic nanospheres are prepared by the following steps: Mix magnetite and γ - ferric oxide, ultrasonically disperse them in the polyvinyl alcohol aqueous solution to obtain a dispersion; add tetraethyl orthosilicate to the dispersion, heat and react, then cool down, add dopamine, adjust the pH to alkaline for reaction, and then carry out magnetic separation and washing to obtain magnetic nanospheres.

7. An application process of nitrile hydratase catalysis for efficiently preparing acrylamide according to claim 6, characterized in that: In the step (2), cross-linking is carried out in the calcium chloride solution at 3 - 5 °C for 12 - 18 min, in the citric acid solution at 28 - 32 °C for 15 - 25 min, and in the polyethyleneimine solution at 3 - 5 °C for 1.5 - 2.5 h.

8. The catalytic application process of nitrile hydratase for efficiently preparing acrylamide according to claim 1, wherein: In the step (3), the initial concentration of acrylonitrile is 3 - 5%, the addition amount of the nitrile hydratase catalyst is 10 - 15 g / L, the initial temperature of the reaction solution is 16 - 20 °C, stir the reaction at a speed of 200 - 300 rpm for 0.5 - 1 h, raise the temperature to 30 - 32 °C and react for 2 - 3 h. After the acrylamide concentration reaches 15%, add acrylonitrile to maintain the acrylonitrile concentration at 4 - 6%. After the propionamide concentration reaches 30 - 40%, lower the temperature to 24 - 26 °C and react for 2 - 3 h. During the hydration reaction, control the pH at 7.2 - 7.

8.

9. The catalytic application process of nitrile hydratase for efficiently preparing acrylamide according to claim 8, characterized in that: Hydroquinone and copper sulfate are added to the reaction solution in the step (3).

10. The catalytic application process of nitrile hydratase for efficiently preparing acrylamide according to claim 1, wherein: In the step (4), separation is carried out in a strong magnetic field of 2-3 T for 5-10 min, and the flow rate of the product mixture is 0.5-0.8 m 3 / h. Then, washing and separation are carried out at an intensity of 0.5-1 T for 10-15 min, and the washing flow rate is 0.8-1.0 m 3 / h.

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