Magnetic immobilized enzyme for nicotinic acid synthesis and preparation method thereof

Abstract

The application discloses a kind of magnetic immobilized enzyme for nicotinic acid synthesis and preparation method thereof, belong to magnetic immobilized enzyme technical field, first with pyridine-3-carboxylic acid and benzyl bromide as raw material, by nucleophilic substitution 1-benzyl-3-carboxyl pyridine bromide is prepared, it is grafted to chitosan by amidation reaction, obtain benzyl pyridine chitosan, then chemical coprecipitation method is used to prepare benzyl pyridine chitosan@Fe3O4 Magnetic particles, finally through glutaraldehyde covalent crosslinking fixation nitrile hydrolase, obtain the magnetic immobilized enzyme for nicotinic acid synthesis;The application improves the positive electric property and biocompatibility of carrier by introducing benzyl pyridine quaternary ammonium salt group, combined with the quick separation and recovery characteristics of magnetic carrier, cooperatively solve the problems of poor stability of free enzyme, difficult to recycle, significantly improve the catalytic efficiency and reusability of enzyme in the process of nicotinic acid synthesis, suitable for the green biological catalytic production field of pharmaceutical intermediate nicotinic acid.

Description

Technical Field

[0001] This invention belongs to the field of magnetic immobilized enzyme technology, specifically a magnetic immobilized enzyme for nicotinic acid synthesis and its preparation method. Background Technology

[0002] Niacin (vitamin B3) is an important pharmaceutical, feed, and food additive. Currently, its industrial production mainly relies on chemical synthesis, but this method suffers from problems such as harsh reaction conditions, numerous byproducts, and severe environmental pollution. In recent years, biocatalytic synthesis of niacin using 3-cyanopyridine as a substrate via nitrile hydrolase or nitrile hydratase / amidase has emerged as a highly promising green alternative process due to its mild reaction conditions, high selectivity, and environmental friendliness.

[0003] Free enzymes face bottlenecks in industrial applications, including difficulty in recovery, poor stability, and limited reusability. While enzyme immobilization technology, which fixes enzymes onto insoluble carriers, allows for enzyme recovery, separation relies on centrifugation or filtration, which is cumbersome and prone to enzyme loss, thus limiting industrial applications. To improve separation efficiency, Chinese patent application CN107746842A discloses a method for preparing enzymes immobilized on magnetic cellulose microspheres. This method physically mixes magnetic iron oxide nanoparticles into a cellulose gel network, solving the problems of cumbersome separation operations, easy enzyme loss, and low recovery efficiency associated with traditional carriers.

[0004] However, in this scheme, the Fe3O4 magnetic nanoparticles are physically mixed in a cellulose gel network, lacking a dense chemical bond and protective layer with the carrier framework. When this carrier is applied to the biosynthesis system of nicotinic acid, continuous acid production during the reaction leads to a decrease in the pH value of the system. + The permeation of the gel network directly corrodes the Fe3O4 magnetic core, causing iron ions to dissolve. The dissolved iron ions poison the enzyme's active site, accelerate enzyme activity loss, and destroy the magnetic core structure, leading to magnetic attenuation and carrier failure. This shortens the lifespan of the immobilized enzyme and reduces the enzyme preparation recovery efficiency, thus limiting its industrial application in strongly acidic systems such as nicotinic acid synthesis. Summary of the Invention

[0005] The purpose of this invention is to provide a magnetically immobilized enzyme for nicotinic acid synthesis and its preparation method. By introducing benzylpyridine quaternary ammonium salt groups to enhance the positive charge and biocompatibility of the carrier, and combining the rapid separation and recovery characteristics of the magnetic carrier, the problem of poor stability and difficulty in recycling and reuse of free enzymes is synergistically solved, significantly improving the catalytic efficiency and reusability of the enzyme in the nicotinic acid synthesis process. It is applicable to the green biocatalytic production of nicotinic acid, a pharmaceutical intermediate.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] A method for preparing a magnetically immobilized enzyme for nicotinic acid synthesis includes the following steps:

[0008] Step 1: Using pyridine-3-carboxylic acid and benzyl bromide as raw materials, 1-benzyl-3-carboxypyridine bromide is prepared by nucleophilic substitution reaction, and then chitosan is grafted by amidation reaction to obtain benzylpyridine chitosan.

[0009] Step 2: Using a chemical co-precipitation method, Fe3O4 magnetic nanoparticles were generated in situ under alkaline conditions using ferrous ammonium sulfate hexahydrate and ferric ammonium sulfate dodecahydrate as iron sources. Simultaneously, the nanoparticles were coated with benzylpyridine chitosan to obtain benzylpyridine chitosan@Fe3O4 magnetic particles.

[0010] Step 3: Nitrile hydrolase was immobilized on the surface of benzylpyridine chitosan@Fe3O4 magnetic particles by glutaraldehyde covalent cross-linking to obtain a magnetically immobilized enzyme for nicotinic acid synthesis.

[0011] Furthermore, the specific preparation steps of the magnetically immobilized enzyme used for nicotinic acid synthesis are as follows:

[0012] Benzylpyridine chitosan@Fe3O4 magnetic particles and phosphate buffer were added to a reaction vessel and sonicated for 8-12 min to ensure complete dispersion. A 5% (v / v) glutaraldehyde solution was added to the vessel and activated at 23-27℃ and 100-200 r / min for 50-70 min. After magnetic separation and washing, a nitrile hydrolase solution was added and slowly stirred at 3-5℃ for 11-13 h for adsorption. The residual aldehyde groups were blocked with 0.1 M glycine. After magnetic separation and washing, the mixture was vacuum dried at 4℃ for 12 h to obtain the magnetically immobilized enzyme for nicotinic acid synthesis.

[0013] Furthermore, the ratio of benzylpyridine chitosan@Fe3O4 magnetic particles, phosphate buffer, glutaraldehyde solution, and nitrile hydrolase solution is 1.8-2.2g: 90-110mL: 1.8-2.2mL: 15-25mL.

[0014] Furthermore, the nitrile hydrolase solution is prepared by mixing nitrile hydrolase and 0.1M phosphate buffer at a ratio of 300-350 mg: 15-25 mL.

[0015] Furthermore, the specific preparation steps of benzylpyridine chitosan@Fe3O4 magnetic particles are as follows:

[0016] Ferrous ammonium sulfate hexahydrate, ferric ammonium sulfate dodecahydrate, and deionized water were added to a reaction vessel and stirred until dissolved. Then, benzylpyridine chitosan was added to the vessel and ultrasonically stirred for 20-40 min. The pH was adjusted to 10 with 6 mol / L ammonia water. After the pH stabilized, the reaction was continued at 45-55℃ and 200-300 r / min for 50-70 min. After the reaction was completed, the mixture was cooled to room temperature and allowed to stand for 10-15 min. The magnetic product was adsorbed with a magnet, the supernatant was discarded, and the mixture was washed with deionized water until the last wash solution was neutral. The mixture was then vacuum dried at 60℃ to constant weight to obtain benzylpyridine chitosan@Fe3O4 magnetic particles.

[0017] Furthermore, the ratio of ferrous ammonium sulfate hexahydrate, ferric ammonium sulfate dodecahydrate, deionized water, and benzylpyridine chitosan is 6.5-7g: 10-11g: 450-550mL: 3.5-4.5g.

[0018] Furthermore, the specific preparation steps of benzylpyridine chitosan are as follows:

[0019] Chitosan and a 2% (v / v) acetic acid solution were added to a reaction vessel and stirred at 23-27°C and 250-350 rpm until dissolved. The activated quaternary ammonium salt solution was slowly added dropwise to the reaction vessel and stirred under the same conditions for 22-26 hours. After the reaction was completed, the product was transferred to a dialysis bag and dialyzed with deionized water for 3 days. Finally, it was freeze-dried for 48 hours to obtain benzylpyridine chitosan.

[0020] Furthermore, the ratio of chitosan, acetic acid solution, and activated quaternary ammonium salt solution is 6.2-6.8g: 950-1050mL: 380-420mL.

[0021] Furthermore, the activated quaternary ammonium salt solution is obtained by mixing 1-benzyl-3-carboxypyridine bromide, deionized water, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in a ratio of 23-25g:380-420mL:15-17g:9-11g.

[0022] Furthermore, the specific preparation steps of 1-benzyl-3-carboxypyridine bromide are as follows:

[0023] Pyridine-3-carboxylic acid, benzyl bromide and acetonitrile were added to a reaction vessel and refluxed at 80-85℃ and 300r / min for 12-16h. After cooling, diethyl ether was added to precipitate the product. The product was filtered and the filter cake was washed with diethyl ether to obtain 1-benzyl-3-carboxypyridine bromide.

[0024] Furthermore, the ratio of pyridine-3-carboxylic acid, benzyl bromide, and acetonitrile is 24-26 g: 35-37 g: 480-520 mL.

[0025] The beneficial effects of this invention are:

[0026] 1. This invention utilizes benzylpyridine chitosan to in-situ coat Fe3O4 magnetic cores, forming a relatively dense, acid-resistant protective layer on the core surface. This improves upon the lack of chemical bonding between the magnetic core and the carrier framework under physical mixing methods, and helps resist the H+ in the nicotinic acid synthesis system. + The penetration corrosion reduces the dissolution of iron ions, thereby mitigating the impact of iron ions on the enzyme's active center, maintaining the relative stability of the magnetic core structure, which helps extend the lifespan of the immobilized enzyme, improves the enzyme preparation recovery efficiency, and better adapts to the industrial application of strongly acidic systems.

[0027] 2. The benzylpyridine chitosan prepared in this invention, by introducing benzylpyridine quaternary ammonium salt groups onto the chitosan molecular chain, can endow the carrier with certain antibacterial properties, which is beneficial for inhibiting the growth of miscellaneous bacteria and reducing the risk of biological contamination during long-term catalysis. The large number of amino groups retained in its molecular chain provides more cross-linking sites for subsequent enzyme immobilization. Simultaneously, the quaternary ammonium salt groups and hydrophobic benzyl groups can synergistically form a hydrophilic-hydrophobic microenvironment, which helps improve the dispersibility of the carrier in the aqueous phase and has a certain enrichment effect on the hydrophobic substrate 3-cyanopyridine, which is beneficial for alleviating product inhibition and improving catalytic efficiency. The permanent positive charge of the pyridine quaternary ammonium salt can block H+ through electrostatic repulsion in an acidic environment. + Osmosis forms a local charge barrier, which buffers and protects enzyme molecules, and helps to broaden the pH range of the process operation.

[0028] 3. This invention employs a chemical co-precipitation method to achieve in-situ uniform coating of Fe3O4 magnetic cores with benzylpyridine chitosan. The chitosan molecular chains and the surface of the magnetic core form a core-shell structure through coordination bonds and electrostatic interactions, providing both physical and chemical protection for the magnetic core. This helps to block H2O from the nicotinic acid synthesis system. + This reduces erosion, iron ion dissolution, magnetic core corrosion, and magnetic attenuation. Simultaneously, the magnetic particles possess superparamagnetism and good dispersibility, enabling rapid separation and recovery of immobilized enzymes under an applied magnetic field, simplifying downstream operations and reducing losses.

[0029] 4. This invention uses glutaraldehyde covalent cross-linking to immobilize nitrile hydrolase. Relying on the abundant amino sites provided by benzylpyridine chitosan, the enzyme and carrier are stably bound under mild conditions, which helps to reduce enzyme molecule shedding and better preserve the enzyme's catalytic activity, thereby improving the relative enzyme activity retention rate and reusability stability of the immobilized enzyme. Combined with an acid-resistant protective layer and antibacterial effect, the immobilized enzyme can maintain a relatively stable operating state in the acidic nicotinic acid synthesis system, extending the enzyme preparation's service life and making it more suitable for long-term reuse in industrial applications. Detailed Implementation

[0030] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1: A magnetically immobilized enzyme for nicotinic acid synthesis, prepared by the following steps:

[0032] S1: 25g of pyridine-3-carboxylic acid, 36g of benzyl bromide and 500mL of acetonitrile were added to the reaction vessel and refluxed at 82℃ and 300r / min for 14h. After cooling, ether was added to precipitate the product. The product was filtered and the filter cake was washed with ether to obtain 1-benzyl-3-carboxypyridine bromide.

[0033] Based on nucleophilic substitution, the pyridine nitrogen atom of pyridine-3-carboxylic acid reacts with the benzyl carbon atom of benzyl bromide to generate 1-benzyl-3-carboxypyridine bromide.

[0034] S2: Add 24g of 1-benzyl-3-carboxypyridine bromide and 400mL of deionized water to a reaction vessel, add 16g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 10g of N-hydroxysuccinimide (NHS), and activate for 1h at a temperature of 25℃ and a rotation speed of 200r / min to obtain an activated quaternary ammonium salt solution.

[0035] 6.48 g of chitosan and 1000 mL of 2% acetic acid solution were added to a reaction vessel and stirred at 25 °C and 300 r / min until completely dissolved. 400 mL of activated quaternary ammonium salt solution was slowly added dropwise to the reaction vessel and stirred for 24 h under the same conditions. After the reaction was completed, the product was transferred to a dialysis bag (molecular weight cutoff 8000), dialyzed with deionized water for 3 days, and finally freeze-dried for 48 h to obtain benzylpyridine chitosan.

[0036] Through an EDC / NHS activation condensation reaction, using EDC·HCl as the condensing agent and NHS as the activating agent, in a weakly acidic aqueous environment, the carboxyl group of 1-benzyl-3-carboxypyridine bromide undergoes an amidation reaction with the amino group on the chitosan molecular chain, introducing a benzylpyridine quaternary ammonium salt structure. After purification, benzylpyridine chitosan, which combines the biocompatibility of chitosan with the strong antibacterial activity of quaternary ammonium salt, is obtained.

[0037] S3: Add 6.8g of ferrous ammonium sulfate hexahydrate, 10.4g of ferric ammonium sulfate dodecahydrate, and 500mL of deionized water to a reaction vessel and stir until completely dissolved. Then add 4g of benzylpyridine chitosan to the vessel and sonicate for 0.5h. Adjust the pH to 10 with 6mol / L ammonia water. After the pH stabilizes, continue the reaction for 1h at 50℃ and 200-300r / min. After the reaction is complete, cool to room temperature and let stand for 10-15min. Adsorb the magnetic product with a magnet, discard the supernatant, and wash with deionized water until the last wash solution is neutral. Vacuum dry at 60℃ to constant weight to obtain benzylpyridine chitosan@Fe3O4 magnetic particles.

[0038] Magnetic Fe3O4 nanoparticles were generated under alkaline conditions using ferrous ammonium sulfate hexahydrate and ferric ammonium sulfate dodecahydrate as iron sources via chemical coprecipitation. The magnetic particles were uniformly coated with chitosan by electrostatic adsorption and coordination between the pyridine quaternary ammonium salt cations in benzylpyridine chitosan molecules and the Fe3O4 surface under stirring at 50°C. After magnetic separation washing and vacuum drying, benzylpyridine chitosan@Fe3O4 magnetic particles with both superparamagnetic responsiveness and an amphiphilic coating were obtained.

[0039] S4: Dissolve 330 mg of nitrile hydrolase in 20 mL of 0.1 M phosphate buffer (pH 7) to obtain nitrile hydrolase solution; add 2 g of benzylpyridine chitosan@Fe3O4 magnetic particles and 100 mL of phosphate buffer to the reaction vessel, sonicate for 10 min to fully disperse them, add 2 mL of 5% (V / V) glutaraldehyde solution to the vessel, activate for 1 h at 25 °C and 150 r / min, after magnetic separation and washing, add 20 mL of nitrile hydrolase solution, slowly stir and adsorb for 12 h at 4 °C, block the residual aldehyde group with 0.1 M glycine, after magnetic separation and washing, vacuum dry at 4 °C for 12 h to obtain magnetically immobilized enzyme for nicotinic acid synthesis.

[0040] Example 2: A magnetically immobilized enzyme for nicotinic acid synthesis, prepared by the following steps:

[0041] S1: 24g of pyridine-3-carboxylic acid, 35g of benzyl bromide and 480mL of acetonitrile were added to the reaction vessel and refluxed at 80℃ and 300r / min for 12h. After cooling, ether was added to precipitate the product. The product was filtered and the filter cake was washed with ether to obtain 1-benzyl-3-carboxypyridine bromide.

[0042] S2: Add 23g of 1-benzyl-3-carboxypyridine bromide and 380mL of deionized water to a reaction vessel, add 15g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 9g of N-hydroxysuccinimide (NHS), and activate for 50min at 23℃ and 180r / min to obtain an activated quaternary ammonium salt solution.

[0043] 6.2 g of chitosan and 950 mL of 2% acetic acid solution were added to a reaction vessel and stirred at 23 °C and 250 r / min until completely dissolved. 400 mL of activated quaternary ammonium salt solution was slowly added dropwise to the reaction vessel and stirred for 22 h under the same conditions. After the reaction was completed, the product was transferred to a dialysis bag (molecular weight cutoff 8000), dialyzed with deionized water for 3 days, and finally freeze-dried for 48 h to obtain benzylpyridine chitosan.

[0044] S3: Add 6.5g of ferrous ammonium sulfate hexahydrate, 10g of ferric ammonium sulfate dodecahydrate, and 450mL of deionized water to the reaction vessel and stir until completely dissolved. Then add 3.5g of benzylpyridine chitosan to the vessel and sonicate for 20min. Adjust the pH to 10 with 6mol / L ammonia water. After the pH stabilizes, continue the reaction for 50min at 45℃ and 200r / min. After the reaction is complete, cool to room temperature and let stand for 10min. Adsorb the magnetic product with a magnet, discard the supernatant, and wash with deionized water until the last wash solution is neutral. Vacuum dry at 60℃ to constant weight to obtain benzylpyridine chitosan@Fe3O4 magnetic particles.

[0045] S4: Dissolve 300 mg of nitrile hydrolase in 15 mL of 0.1 M phosphate buffer (pH 7) to obtain nitrile hydrolase solution; add 1.8 g of benzylpyridine chitosan@Fe3O4 magnetic particles and 90 mL of phosphate buffer (pH 7) to the reaction vessel, sonicate for 8 min to fully disperse, add 1.8 mL of 5% (V / V) glutaraldehyde solution to the vessel, activate for 50 min at 23℃ and 100 r / min, after magnetic separation and washing, add 15 mL of nitrile hydrolase solution, slowly stir and adsorb for 11 h at 3℃, block the residual aldehyde group with 0.1 M glycine, after magnetic separation and washing, vacuum dry at 4℃ for 12 h to obtain magnetically immobilized enzyme for nicotinic acid synthesis.

[0046] Example 3: A magnetically immobilized enzyme for nicotinic acid synthesis, prepared by the following steps:

[0047] S1: 26g of pyridine-3-carboxylic acid, 37g of benzyl bromide and 520mL of acetonitrile were added to the reaction vessel and refluxed at 85℃ and 300r / min for 16h. After cooling, ether was added to precipitate the product. The product was filtered and the filter cake was washed with ether to obtain 1-benzyl-3-carboxypyridine bromide.

[0048] S2: Add 25g of 1-benzyl-3-carboxypyridine bromide and 420mL of deionized water to a reaction vessel, add 17g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 11g of N-hydroxysuccinimide (NHS), and activate for 70min at a temperature of 27℃ and a rotation speed of 220r / min to obtain an activated quaternary ammonium salt solution.

[0049] 6.8 g of chitosan and 1050 mL of 2% acetic acid solution were added to a reaction vessel and stirred at 27 °C and 350 r / min until completely dissolved. 400 mL of activated quaternary ammonium salt solution was slowly added dropwise to the reaction vessel and stirred for 26 h under the same conditions. After the reaction was completed, the product was transferred to a dialysis bag (molecular weight cutoff 8000), dialyzed with deionized water for 3 days, and finally freeze-dried for 48 h to obtain benzylpyridine chitosan.

[0050] S3: Add 7g of ferrous ammonium sulfate hexahydrate, 11g of ferric ammonium sulfate dodecahydrate, and 550mL of deionized water to the reaction vessel and stir until completely dissolved. Then add 4.5g of benzylpyridine chitosan to the vessel and sonicate for 40min. Adjust the pH to 10 with 6mol / L ammonia water. After the pH stabilizes, continue the reaction for 70min at 55℃ and 300r / min. After the reaction is completed, cool to room temperature and let stand for 15min. Adsorb the magnetic product with a magnet, discard the supernatant, wash with deionized water until the last wash solution is neutral, and vacuum dry at 60℃ to constant weight to obtain benzylpyridine chitosan@Fe3O4 magnetic particles.

[0051] S4: Dissolve 350 mg of nitrile hydrolase in 25 mL of 0.1 M phosphate buffer (pH 7) to obtain nitrile hydrolase solution; add 2.2 g of benzylpyridine chitosan@Fe3O4 magnetic particles and 110 mL of phosphate buffer (pH 7) to the reaction vessel, sonicate for 12 min to ensure complete dispersion, add 2.2 mL of 5% (V / V) glutaraldehyde solution to the vessel, activate for 70 min at 27 °C and 200 r / min, after magnetic separation and washing, add 25 mL of nitrile hydrolase solution, slowly stir and adsorb for 13 h at 5 °C, block residual aldehyde groups with 0.1 M glycine, after magnetic separation and washing, vacuum dry at 4 °C for 12 h to obtain magnetically immobilized enzyme for nicotinic acid synthesis.

[0052] All materials used in Examples 1-3 of this application are commercially available. Nicotinic acid (99.5%) and acetonitrile (HPLC, ≥99.9%) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; benzyl bromide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) were purchased from Anaiji Chemical Reagent Co., Ltd. (Shanghai, China); ferrous ammonium sulfate hexahydrate (99%) and ferric ammonium sulfate dodecahydrate (99%) were purchased from Shanghai Dibai Biotechnology Co., Ltd.; nitrile hydrolase (activity ≥10U / mg) was purchased from Wuhan Weissman Bioengineering Co., Ltd.; glutaraldehyde (AR, 50%) and chitosan (degree of deacetylation ≥95%, viscosity 100-200mPa·s) were purchased from Shanghai Aladdin Biochemical Co., Ltd.

[0053] Comparative Example 1: Based on Example 1, steps S1 and S2 for the preparation of benzylpyridine chitosan were omitted. In step S3, benzylpyridine chitosan was replaced with an equal amount of unmodified chitosan. All other steps and parameters remained unchanged to obtain a magnetically immobilized enzyme.

[0054] Comparative Example 2: Based on Example 1, the addition of glutaraldehyde and glycine blocking were omitted in step S4. The nitrile hydrolase was loaded onto the benzylpyridine chitosan@Fe3O4 magnetic support only by physical adsorption. All other steps and parameters remained unchanged to obtain the magnetically immobilized enzyme.

[0055] The performance of the magnetically immobilized enzymes prepared in Examples 1-3 and Comparative Examples 1-2 was tested. For enzyme activity determination, 3-cyanopyridine was used as the substrate, and the catalytic reaction was carried out at pH 7.0 and 30°C. 0.1 g of the magnetically immobilized enzyme was added to 50 mL of 3-cyanopyridine solution, and the mixture was shaken at 30°C for 10 min. After centrifugation, the concentration of nicotinic acid in the supernatant was determined using high-performance liquid chromatography (HPLC), and the enzyme activity (U / g) was calculated. The relative enzyme activity retention rate was determined based on the total activity of an equal volume of free enzyme, calculated using the formula: Relative enzyme activity retention rate (%) = (Total immobilized enzyme activity / Total free enzyme activity) × 100%. The result is expressed as a percentage (%). A higher value indicates better catalytic efficiency and enzyme activity retention of the magnetically immobilized enzyme, and a smaller impact of the immobilization process on enzyme activity.

[0056] The stability assay for repeated use involved magnetically immobilized enzyme undergoing one catalytic reaction, followed by magnetic separation and recovery using an external magnet. The supernatant was discarded, and 5 mL of 0.1 M phosphate buffer (pH 7) was added. The mixture was gently agitated at room temperature for 1 min, and magnetic separation was performed again using an external magnet. The supernatant was discarded. This washing process was repeated twice (a total of three washes) to remove residual substrate, product, and any potentially detached free enzyme molecules from the carrier surface. After washing, the magnetically immobilized enzyme was re-added to a fresh substrate solution (50 mL, 10 mM) of equal volume and concentration as the first reaction. In 3-cyanopyridine phosphate buffer (pH 7), the next round of catalytic reaction was carried out at a temperature of 30℃ and a rotation speed of 150 r / min. This operation was repeated 10 times. After each reaction, samples were taken and centrifuged. The concentration of nicotinic acid in the supernatant was determined by high performance liquid chromatography (HPLC). The enzyme activity (U / g) of each reaction was calculated. With the enzyme activity of the first reaction as 100%, the relative enzyme activity (%) of the nth reaction was calculated according to the formula: (nth reaction enzyme activity / first reaction enzyme activity) × 100%. The results are expressed as percentages (%). The higher the value, the better the operational stability and reusability of the corresponding magnetically immobilized enzyme, which is more in line with the application requirements of long-term reuse in industrial applications.

[0057] The results are shown in Table 1:

[0058] Table 1 Performance test results of each magnetically immobilized enzyme

[0059] Project Group Enzyme activity (U / g) Relative enzyme activity retention rate (%) Relative enzyme activity (%) after 10 repeated uses Example 1 285 89.2 78.5 Example 2 278 87.8 76.3 Example 3 292 91.5 81.2 Comparative Example 1 221 70.3 42.7 Comparative Example 2 236 75.1 31.9

[0060] As shown in Table 1, the magnetically immobilized enzymes for nicotinic acid synthesis prepared in Examples 1-3 of this invention exhibit superior performance in terms of enzyme activity, relative enzyme activity retention rate, and reusability. This is achieved through a preparation process involving the construction of a chitosan-modified benzylpyridine quaternary ammonium salt-coated Fe3O4 magnetic core and glutaraldehyde covalently cross-linked immobilized nitrile hydrolase. This process helps to improve the problems of poor acid resistance of magnetic carriers, easy corrosion of magnetic cores, rapid enzyme activity loss, and poor recycling and reuse in existing technologies. To a certain extent, it improves the catalytic efficiency, operational stability, and reusability of immobilized enzymes, making them more suitable for the industrial application requirements of green biocatalytic synthesis of nicotinic acid.

[0061] Comparative Example 1 used ordinary chitosan to coat magnetic particles to immobilize enzymes, lacking the benzylpyridine quaternary ammonium salt modified chitosan component. Its overall performance was significantly inferior to that of the Example, with a marked difference in relative enzyme activity retention and reusability stability. This may be related to the absence of the benzylpyridine quaternary ammonium salt group modification and dense coating effect, making the magnetic core easily absorbed by H+ in the acidic nicotinic acid synthesis system. +The corrosion leads to the dissolution of iron ions, which in turn affects the enzyme active site, resulting in an accelerated decline in enzyme activity. This result indicates that benzylpyridine chitosan can simultaneously enhance the positive charge and biocompatibility of the carrier and construct an acid-resistant protective layer, making it an important component for achieving a synergistic effect of high enzyme activity retention and good acid resistance.

[0062] Comparative Example 2 used physical adsorption to immobilize the enzyme, omitting the glutaraldehyde covalent cross-linking step. It relied solely on physical adsorption to load the nitrile hydrolase, and its reusability stability was relatively poor among the test groups. This may be related to the lack of covalent cross-linking chemical bonding, which makes the enzyme molecules more likely to detach from the carrier surface during multiple catalysis and recovery processes, leading to rapid degradation of enzyme activity. This result indicates that glutaraldehyde covalent cross-linking is a key step in achieving stable binding between the enzyme and the carrier and improving the long-term operational stability of the immobilized enzyme, playing an important role in obtaining reusable enzyme preparations.

[0063] It should be noted that, in this document, terms such as “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0064] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention.

Claims

1. A method for preparing a magnetically immobilized enzyme for nicotinic acid synthesis, characterized in that, Prepared by the following steps: Step 1: Using pyridine-3-carboxylic acid and benzyl bromide as raw materials, 1-benzyl-3-carboxypyridine bromide is prepared by nucleophilic substitution reaction, and then chitosan is grafted by amidation reaction to obtain benzylpyridine chitosan; Step 2: Using a chemical co-precipitation method, Fe3O4 magnetic nanoparticles were generated in situ under alkaline conditions using ferrous ammonium sulfate hexahydrate and ferric ammonium sulfate dodecahydrate as iron sources. Simultaneously, the nanoparticles were coated with benzylpyridine chitosan to obtain benzylpyridine chitosan@Fe3O4 magnetic particles. Step 3: Nitrile hydrolase was immobilized on the surface of benzylpyridine chitosan@Fe3O4 magnetic particles by glutaraldehyde covalent cross-linking to obtain a magnetically immobilized enzyme for nicotinic acid synthesis.

2. The method for preparing a magnetically immobilized enzyme for nicotinic acid synthesis according to claim 1, characterized in that, The specific preparation steps of the magnetically immobilized enzyme used for nicotinic acid synthesis are as follows: Benzylpyridine chitosan@Fe3O4 magnetic particles and phosphate buffer were added to a reaction vessel and sonicated for 8-12 min to ensure complete dispersion. A 5% (v / v) glutaraldehyde solution was added to the vessel and activated at 23-27℃ and 100-200 r / min for 50-70 min. After magnetic separation and washing, a nitrile hydrolase solution was added and slowly stirred at 3-5℃ for 11-13 h to adsorb the enzyme. The residual aldehyde groups were blocked with 0.1 M glycine. After magnetic separation and washing, the enzyme was vacuum dried at 4℃ for 12 h to obtain the magnetically immobilized enzyme for nicotinic acid synthesis. The ratio of the benzylpyridine chitosan@Fe3O4 magnetic particles, phosphate buffer, glutaraldehyde solution, and nitrile hydrolase solution is 1.8-2.2g: 90-110mL: 1.8-2.2mL: 15-25mL.

3. The method for preparing a magnetically immobilized enzyme for nicotinic acid synthesis according to claim 2, characterized in that, The nitrile hydrolase solution is prepared by mixing nitrile hydrolase and 0.1M phosphate buffer at a ratio of 300-350 mg: 15-25 mL.

4. The method for preparing a magnetically immobilized enzyme for nicotinic acid synthesis according to claim 2, characterized in that, The specific preparation steps of the benzylpyridine chitosan@Fe3O4 magnetic particles are as follows: Ferrous ammonium sulfate hexahydrate, ferric ammonium sulfate dodecahydrate, and deionized water were added to a reaction vessel and stirred until dissolved. Then, benzylpyridine chitosan was added to the vessel and ultrasonically stirred for 20-40 min. The pH was adjusted to 10 with 6 mol / L ammonia water. After the pH stabilized, the reaction was continued at 45-55℃ and 200-300 r / min for 50-70 min. After the reaction was completed, the mixture was cooled to room temperature and allowed to stand for 10-15 min. The magnetic product was adsorbed with a magnet, the supernatant was discarded, and the mixture was washed with deionized water until the last wash solution was neutral. The mixture was then vacuum dried at 60℃ to constant weight to obtain benzylpyridine chitosan@Fe3O4 magnetic particles.

5. The method for preparing a magnetically immobilized enzyme for nicotinic acid synthesis according to claim 4, characterized in that, The ratio of ferrous ammonium sulfate hexahydrate, ferric ammonium sulfate dodecahydrate, deionized water, and benzylpyridine chitosan is 6.5-7g: 10-11g: 450-550mL: 3.5-4.5g.

6. The method for preparing a magnetically immobilized enzyme for nicotinic acid synthesis according to claim 4, characterized in that, The specific preparation steps of the benzylpyridine chitosan are as follows: Chitosan and a 2% (v / v) acetic acid solution were added to a reaction vessel and stirred at 23-27°C and 250-350 rpm until dissolved. The activated quaternary ammonium salt solution was slowly added dropwise to the reaction vessel and stirred under the same conditions for 22-26 hours. After the reaction was completed, the product was transferred to a dialysis bag and dialyzed with deionized water for 3 days. Finally, it was freeze-dried for 48 hours to obtain benzylpyridine chitosan.

7. The method for preparing a magnetically immobilized enzyme for nicotinic acid synthesis according to claim 6, characterized in that, The ratio of chitosan, acetic acid solution, and activated quaternary ammonium salt solution is 6.2-6.8g: 950-1050mL: 380-420mL.

8. The method for preparing a magnetically immobilized enzyme for nicotinic acid synthesis according to claim 6, characterized in that, The activated quaternary ammonium salt solution is obtained by mixing 1-benzyl-3-carboxypyridine bromide, deionized water, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in a ratio of 23-25g:380-420mL:15-17g:9-11g.

9. The method for preparing a magnetically immobilized enzyme for nicotinic acid synthesis according to claim 6, characterized in that, The specific preparation steps for the 1-benzyl-3-carboxypyridine bromide are as follows: Pyridine-3-carboxylic acid, benzyl bromide and acetonitrile were added to a reaction vessel and refluxed at 80-85℃ and 300r / min for 12-16h. After cooling, diethyl ether was added to precipitate the product. The product was filtered and the filter cake was washed with diethyl ether to obtain 1-benzyl-3-carboxypyridine bromide. The ratio of pyridine-3-carboxylic acid, benzyl bromide, and acetonitrile is 24-26g: 35-37g: 480-520mL.

10. A magnetically immobilized enzyme for nicotinic acid synthesis, characterized in that, It is prepared by the preparation method described in any one of claims 1-9.