Immobilized biological enzyme, preparation method thereof and synthesis method of astaxanthin monoester

Abstract

The invention belongs to the field of astaxanthin synthesis, and particularly relates to an immobilized biological enzyme, a preparation method thereof and a synthesis method of astaxanthin monoester. The preparation method of the immobilized bio-enzyme comprises the following steps: deriving a magnetic carbon carrier, and immobilizing a bio-enzyme liquid on the derived magnetic carbon carrier. The immobilized bio-enzyme obtained by adopting the method provided by the invention can catalyze free astaxanthin to efficiently synthesize astaxanthin monoester, the yield of astaxanthin monoester is relatively high, meanwhile, magnetic loss caused by removal of astaxanthin monoester from a material can be effectively avoided, the problem that the bio-enzyme is difficult to separate in application is solved, and the obtained immobilized bio-enzyme can be recycled.

Description

Technical Field

[0001] The invention belongs to the field of astaxanthin synthesis, and particularly relates to an immobilized bioenzyme and a preparation method thereof, as well as a synthesis method of astaxanthin monoester. Background Art

[0002] Astaxanthin (3,3′-dihydroxy-4,4-diketo-β,β′-carotene) is a carotenoid of the xanthophyll class, composed of eight isoprene units, and belongs to the keto-carotenoid class. It is widely found in the biological world, especially in the feathers of shrimp, crab, fish and birds of aquatic animals. In addition, some algae can also produce astaxanthin, such as Haematococcus pluvialis, which is both autotrophic and heterotrophic. At the same time, Phaffia rhodozyma, Rhodotorula glutinosus BF-6, Haemococcus, etc. can also produce astaxanthin. The production of artificially synthesized astaxanthin abroad includes Hoffmann-la roche AG in Switzerland and BASF in Germany.

[0003] Although astaxanthin is a carotenoid, some of its biological effects are much stronger than other carotenoids. Astaxanthin is a non-provitamin carotenoid that cannot be converted into vitamins in animals, but it has extremely strong antioxidant properties, can remove free radicals in the body, regulate and reduce these photochemically caused damage, and has a good therapeutic effect on skin cancer caused by ultraviolet rays. Astaxanthin can also significantly promote the production of lymph node antibodies, especially the production of cell-related antigen antibodies in the body. In food, astaxanthin can not only color, but also effectively preserve freshness, prevent discoloration, taste change, and deterioration. Astaxanthin has a stronger anti-photosensitivity effect than β-carotene. The pharmaceutical and food industries use the antioxidant, anti-inflammatory and immune-promoting effects of astaxanthin as a drug to prevent oxidative tissue damage and prepare health foods. Therefore, astaxanthin has a wide range of application prospects.

[0004] In nature, astaxanthin exists in the form of free astaxanthin or astaxanthin fatty acid esters (monoesters or diesters) alone or in a mixture. Most natural astaxanthin exists in the form of astaxanthin esters. These astaxanthin esters are usually long-chain fatty acid esters, including saturated fatty acids such as palmitic acid and stearic acid, and unsaturated fatty acids such as oleic acid, linoleic acid, linolenic acid and docosahexaenoic acid.

[0005] Astaxanthin prepared by microbial fermentation is mainly in free state, but the free astaxanthin has hydrophobicity due to the presence of polyisoprene, which reduces its bioavailability and applicability. At the same time, since free astaxanthin has conjugated double bonds, carbon groups and hydroxyls, it has a high electronic effect, which makes it quite unstable when exposed to air, high temperature and light, thereby greatly reducing its activity and affecting the use effect. Therefore, during feed processing and storage, the pigment is easily degraded to a large extent. If astaxanthin can be converted into astaxanthin ester, it not only has the activity of astaxanthin, but also has new functions due to the introduction of new groups, such as improved hydrophilicity and hydrophobicity, easy digestion and absorption, etc.

[0006] At present, astaxanthin esters are mainly synthesized by chemical methods, using organic bases such as alkali metal alkoxides as catalysts and acyl chlorides as acylating agents. The reaction process is complex and polluting. In the reaction process, astaxanthin is easily isomerized or degraded by heat and oxidation, thereby reducing the reaction yield. The separation and purification of the product is complex and the biological activity of the product is not high. Compared with the chemical method, the enzyme catalysis method for synthesizing astaxanthin esters has the advantages of mild reaction conditions, high catalytic efficiency, and strong catalytic specificity.

[0007] CN104513844A discloses a method for synthesizing astaxanthin succinate using Phaffia rhodozyma as a raw material and lipase catalysis, separation and reaction coupling. The raw material used in the method is the extract concentrate before purification, and yeast lipase and acylating agent (succinic anhydride) are directly added to catalyze esterification under certain conditions to synthesize astaxanthin succinate with good stability. The process uses the extract concentrate with lower purity for esterification, which is easy to have a certain impact on the formation and stability of astaxanthin esters, and the lipase required for esterification catalysis is not modified and immobilized, and the recovery rate is not high, resulting in high cost and unfavorable for scale-up production.

[0008] CN110218308A discloses a method for preparing methoxy polyethylene glycol acetate astaxanthin ester, which uses chemically synthesized free astaxanthin or free astaxanthin from natural sources as the main skeleton, methoxy polyethylene glycol acetic acid as the acyl donor and hydrophilic group, uses a catalyst to catalyze the synthesis reaction, and then purifies to obtain methoxy polyethylene glycol acetate astaxanthin ester. In this method, 4-dimethylaminopyridine (DMAP) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) are used as chemical catalysts, and the catalyst is removed by acid washing, alkaline washing, and brine washing in the later stage. However, the yield of astaxanthin ester prepared by using chemical catalyst is low, and there are safety issues caused by incomplete washing of chemical catalysts, astaxanthin ester is easy to isomerize during the esterification process, has low production capacity, and high cost.

[0009] Furthermore, because astaxanthin is a secondary alcohol, and there is a ketone group on the adjacent position that occupies a larger spatial position, this makes the steric hindrance of astaxanthin in the esterification process larger, and it is difficult to catalyze the esterification synthesis of astaxanthin esters using ordinary biological enzymes alone. In addition, free astaxanthin is easy to synthesize a mixture of astaxanthin monoesters and diesters during the esterification synthesis process. Since astaxanthin diesters are more lipophilic than monoesters, astaxanthin diesters are not as easy to digest and absorb as astaxanthin monoesters. At the same time, astaxanthin monoesters have better DPPH clearance and ABTS clearance rates than astaxanthin diesters. Therefore, there is an urgent need to develop a method for efficiently catalyzing the esterification synthesis of astaxanthin esters, especially astaxanthin monoesters, so that the industrial production of astaxanthin monoesters synthesized from astaxanthin becomes possible. Summary of the invention

[0010] The first object of the present invention is to provide a method for preparing an immobilized bio-enzyme, wherein the immobilized bio-enzyme obtained by the method can efficiently catalyze the esterification synthesis of astaxanthin monoester.

[0011] The second object of the present invention is to provide an immobilized bioenzyme prepared by the above method.

[0012] The third object of the present invention is to provide a method for synthesizing astaxanthin monoester using the above-mentioned immobilized bioenzyme.

[0013] Specifically, the method for preparing the immobilized enzyme provided by the present invention comprises the following steps: S1. Preparation of derived magnetic carbon carrier: an organic carrier, a phosphate and an organosilicon source are subjected to a sol-gel reaction in a hydrochloric acid solution, the obtained gel is subjected to high temperature aging to separate solid particles, the obtained solid particles are then washed with water and dried, and then the obtained silicate solid particles are immersed in an iron salt solution for solid-liquid separation, and then the obtained solid particles are pyrolyzed and carbonized to obtain a derived magnetic carbon carrier; S2. Enzyme immobilization: The derived magnetic carbon carrier is dispersed in a phosphate buffer solution, the pH value of the obtained dispersion is adjusted to 5-8 and then ultrasonically activated, and then the obtained activation solution and the biological enzyme solution are ultrasonically treated so that the biological enzyme solution is adsorbed on the carrier to obtain an immobilized biological enzyme.

[0014] The method for synthesizing astaxanthin monoester provided by the present invention comprises subjecting free astaxanthin to esterification reaction with unsaturated fatty acid in the presence of the above-mentioned immobilized biological enzyme.

[0015] The immobilized bio-enzyme obtained by the method provided by the present invention can catalyze the free astaxanthin to efficiently synthesize astaxanthin monoester, and the yield of astaxanthin monoester is relatively high. Furthermore, the present invention performs magnetic modification on the carbon carrier, embeds the magnetic species into the carrier trunk, and can effectively prepare a porous carbon carrier with stable magnetic modification. Compared with directly connecting the magnetic nanoparticles to the surface of the material through weak interactions, it can effectively avoid removing the magnetic loss caused by removing from the material during use, and solves the problems of limited stability and difficult separation in the application of bio-enzymes at the same time, and the obtained immobilized enzyme can be recycled and reused. In addition, the present invention introduces ultrasonic activation during the assimilation of enzyme fixation, which is not only conducive to the firm connection between the carrier and the enzyme, but also can prevent the carrier from agglomerating during the adsorption of the bio-enzyme, playing a dual role.

[0016] The principle of magnetic attraction of the derived magnetic carbon carrier provided by the present invention is: Fe and Si elements in the Fe-silicate on the derived magnetic carbon carrier obtained by pyrolysis and carbonization are respectively present in the form of Fe3C and silicon sulfide. The etching effect on the porous carbon material framework during the formation of these substances leads to changes in the carrier structure. Among them, the iron salt enters the pore structure of the silicate solid particles during the immersion process, and then undergoes high-temperature pyrolysis and carbonization together with the silicate solid particles. Fe 2+ It may be reduced by C and other substances in the framework of porous carbon materials and penetrate into the carbon framework to form Fe3C. The divalent S element in the iron salt (ferrous sulfate) is reduced and reacts with Si 2+ Combined to form silicon sulfide. Some O elements and C elements in the framework form volatile carbon oxides and volatilize. However, the Fe3C substance generated at this moment makes the prepared carrier magnetic and can be attracted by the magnet. The magnetic effect produced can significantly enhance the catalytic activity of the enzyme in the later stage and improve the recovery rate and reuse rate after use.

[0017] In a preferred embodiment, the preparation method of the immobilized bio-enzyme further includes the step of modifying and cross-linking the immobilized bio-enzyme to obtain an immobilized magnetic carbonized lipase, and the modification and cross-linking process includes: performing a modification reaction on the immobilized bio-enzyme and a modifier, performing a cross-linking reaction on the obtained modified immobilized bio-enzyme and a cross-linking agent, washing and drying the obtained cross-linking reaction product, and the obtained product is an immobilized magnetic carbonized lipase. At this time, by directional modification and fixation of the amino acid residues constituting the catalytic active center of the enzyme, free astaxanthin can be efficiently catalyzed and esterified to synthesize astaxanthin monoester by the enzyme, and the immobilized bio-enzyme obtained in the preparation process becomes difficult to dissolve in the reaction solution due to its immobilization and structural modification, and also promotes the reuse and recovery of the enzyme, improves the original catalytic function of the enzyme, and is more conducive to the efficient esterification of tert-butyl astaxanthin with large steric hindrance and unsaturated fatty acids to synthesize astaxanthin monoester. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a scanning electron microscope image (1 μm) of the solid particles obtained after magnetic attraction in Preparation Example 1; Figure 2 This is a scanning electron microscope image (1 μm) of the derived magnetic carbon carrier obtained in Preparation Example 1; Figure 3 This is a scanning electron micrograph of the immobilized magnetic carbonized lipase obtained in Preparation Example 1 (1 μm); Figure 4 This is a scanning electron micrograph (100 μm) of the immobilized magnetic carbonized lipase obtained in Preparation Example 1. DETAILED DESCRIPTION

[0019] The method for preparing an immobilized biological enzyme provided by the present invention comprises step S1 (ie, preparation of a derivative magnetic carbon carrier) and step S2 (ie, enzyme immobilization).

[0020] In the preparation process of the above-mentioned immobilized bio-enzyme, step S1 specifically includes subjecting the organic carrier, phosphate and organosilicon to a sol-gel reaction in a hydrochloric acid solution, subjecting the obtained gel to high-temperature aging to separate solid particles, then washing the obtained solid particles with water and drying them, then immersing the obtained silicate solid particles in an iron salt solution for solid-liquid separation, and then pyrolyzing and carbonizing the obtained solid particles to obtain a derived magnetic carbon carrier.

[0021] In the preparation process of the above-mentioned immobilized bio-enzyme, the derivative magnetic carbon carrier, as a host material, not only has a high enzyme capacity, but also has good stability and mechanical strength. The derivative magnetic carbon carrier has the characteristics of large specific surface area, high porosity, stable structure, etc., and is extremely suitable for enzyme immobilization. Specifically, the present invention first prepares mesoporous silicate solid particles, and then improves them through pyrolysis carbonization magnetic absorption, which can not only increase their porosity and pore area, but also make them have super strong magnetism.

[0022] In the preparation process of the above-mentioned immobilized bio-enzyme, since the structure of the carbon material mainly depends on covalent bonds, high-temperature pyrolysis carbonization can not only improve the chemical stability of the obtained carbon carrier, but also remove some active agents on the surface of the carbon carrier particles to make the pores of the obtained carbon carrier particles larger. The present invention makes a large-pore derivative magnetic carbon carrier by pyrolysis carbonization after impregnating the synthesized silicate solid particles with iron salts, which can increase the reuse rate of the enzyme. At the same time, a major limitation of the large-scale industrial application of immobilized enzymes is the high cost introduced by their separation and recovery. In this regard, the present invention magnetically modifies the enzyme immobilized carrier and embeds the magnetic species into the carrier backbone, so that a porous carbon material with stable magnetic modification can be effectively obtained. Compared with directly using magnetic nanoparticles through weak interactions, the derivative magnetic carbon carrier obtained by the present invention may avoid being removed from the material during use and causing magnetic loss, and at the same time solves the problems of stability limitation and separation difficulty in the application of immobilized lipase.

[0023] In the preparation process of the immobilized bio-enzyme, in the preparation process of the derivative magnetic carbon carrier mentioned in step S1, the sol-gel reaction method includes stirring and dissolving the organic carrier and phosphate in a hydrochloric acid solution, stirring and mixing the obtained solution with the organosilicon source, and then standing. Wherein, the mass ratio of the organic carrier to the phosphate is preferably 1: (1~10), such as 1: 1, 1: 2, 1: 4, 1: 6, 1: 8, 1: 10 or any value therebetween. The phosphate can be, for example, potassium phosphate and / or sodium phosphate, preferably potassium phosphate (K2PO4). The phosphate added in the sol-gel process forms particles with different morphologies during the standing reaction. Compared with conventional rod-shaped particles, these particles are wider, shorter, and less aggregated. The concentration of the hydrochloric acid solution is preferably 0.1~5 mol / L, such as 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 mol / L or any value therebetween. The amount of the hydrochloric acid solution is preferably 1 to 50 times the total weight of the organic carrier, phosphate and organosilicon source, such as 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 times or any value therebetween. The temperature of the stirring and dissolving is preferably 10°C to 100°C, such as 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, 100°C or any value therebetween. The organic carrier is preferably a triblock copolymer, specifically at least one of SBS (styrene-butadiene-3-chloropropylene copolymer), F127 (polyoxyethylene-polyoxypropylene-polyoxyethylene), and P123 (polyethylene oxide-polypropylene oxide-polyethylene oxide). Examples of the organosilicon source include, but are not limited to, at least one of tetraethoxysilane, tetramethoxysilane, triethylsilane, triethoxysilane, diethoxydimethylsilane and methyltriethoxysilane. The mass ratio of the organosilicon source to the organic carrier is preferably (0.4-0.8):1, such as 0.4:1, 0.45:1, 0.5:1, 0.55:1, 0.6:1, 0.65:1, 0.7:1, 0.75:1, 0.8:1 or any value therebetween. The stirring reaction conditions preferably include a temperature of 10°C-50°C, such as 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C or any value therebetween; and a time of 10min-5h, such as 10min, 20min, 30min, 40min, 50min, 60min, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h or any value therebetween.The standing conditions preferably include a temperature of 10°C to 50°C, such as 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C or any value therebetween; and a time of 1h to 48h, such as 1h, 5h, 10h, 12h, 24h, 36h, 40h, 44h, 48h or any value therebetween.

[0024] In the preparation process of the above-mentioned immobilized bio-enzyme, in the preparation process of the derivative magnetic carbon carrier mentioned in step S1, the high temperature aging conditions preferably include a temperature of 140°C to 160°C, such as 140°C, 145°C, 150°C, 155°C, 160°C or any value therebetween; a pressure of 0.8MPa to 1.2MPa, such as 0.8MPa, 0.9MPa, 1MPa, 1.1MPa, 1.2MPa or any value therebetween; and a time of 10h to 48h, such as 10h, 15h, 20h, 24h, 30h, 36h, 40h, 44h, 48h or any value therebetween. The drying conditions preferably include a temperature of 100°C to 110°C, such as 100°C, 102°C, 104°C, 106°C, 108°C, 110°C or any value therebetween; a time of 6h to 24h, such as 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h or any value therebetween. The iron salt solution is particularly preferably a ferrous sulfate solution. The concentration of the iron salt solution is preferably 0.01mol / L to 1mol / L, such as 0.01, 0.02, 0.05, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1mol / L or any value therebetween. The amount of the iron salt solution is preferably 0.5 to 10 times the weight of the silicate solid particles, such as 0.5, 1, 2, 4, 6, 8, 10 times. The soaking time is preferably 1h~24h, such as 1h, 2h, 5h, 8h, 10h, 12h, 15h, 18h, 20h, 24h or any value therebetween. The conditions for the pyrolysis carbonization preferably include a temperature of 500℃~1000℃, such as 500℃, 600℃, 700℃, 800℃, 900℃, 1000℃ or any value therebetween; the time is 10min~10h, such as 10min, 1h, 2h, 4h, 6h, 8h, 10h or any value therebetween. In addition, the method for separating the solid particles after high temperature aging of the obtained gel can be vacuum filtration. The number of times the solid particles are washed with water is preferably 2~3 times. The solid-liquid separation can be vacuum filtration.

[0025] In the preparation process of the above-mentioned biological enzyme, in the enzyme immobilization process mentioned in step S2, the biological enzyme liquid can be various existing enzyme liquids that can catalyze the esterification reaction of astaxanthin with unsaturated fatty acids to generate astaxanthin monoesters, and is particularly preferably Antarctic Candida lipase liquid. The source of the biological enzyme liquid is not particularly limited, and can be obtained by commercial purchase, or prepared according to various existing methods. In a preferred embodiment, the biological enzyme liquid is prepared according to the following method: the Antarctic Candida lipase liquid is subjected to solid-liquid separation, and the obtained bacterial mud is washed with water and then broken, and the obtained broken wall liquid is the biological enzyme liquid. Among them, the solid-liquid separation method can be selected from at least one of filter press, suction filtration, centrifugal separation and membrane filtration. The purpose of the water washing is to remove the salt on the surface of the bacterial mud. The number of times of the water washing can be reasonably selected according to the actual situation, generally 1 to 5 times. The purpose of the broken wall is to allow the target substance in the cell to be fully released and enhance its enzyme activity. The broken wall is preferably carried out in a high-pressure homogenizer. The working pressure of the high pressure homogenizer is preferably controlled within the range of 500 to 2000 bar, such as 500 bar, 800 bar, 1000 bar, 1200 bar, 1500 bar, 1800 bar, 2000 bar or any value therebetween.

[0026] In the preparation process of the above-mentioned biological enzyme, in the enzyme immobilization process mentioned in step S2, the concentration of the phosphate buffer solution is preferably 0.01mol / L~10mol / L, such as 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10mol / L or any value therebetween. The dosage ratio of the derived magnetic carbon carrier to the phosphate buffer solution is preferably 1g: (0.5~50)mL. Specifically, based on the dosage of the derived magnetic carbon carrier as 1g, the dosage of the phosphate buffer solution can be 0.5, 1, 2, 5, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50mL. The conditions for the ultrasonic activation preferably include a temperature of 20°C to 50°C, such as 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C or any value therebetween; a time of 1 min to 60 min, such as 1 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min or any value therebetween. The amount of the bioenzyme solution added is preferably 0.1 to 1 times the volume of the phosphate buffer, such as 0.1, 0.2, 0.4, 0.6, 0.8, 1 times or any value therebetween. The conditions for the ultrasonic treatment preferably include a temperature of 20°C to 70°C, such as 20°C, 30°C, 40°C, 50°C, 60°C, 70°C or any value therebetween; and a time of 1h to 40h, such as 1h, 5h, 10h, 15h, 20h, 25h, 30h, 35h, 40h, 45h, 50h or any value therebetween.

[0027] In a preferred embodiment, the preparation method of the immobilized bio-enzyme further includes the step of modifying and cross-linking the immobilized bio-enzyme to obtain an immobilized magnetic carbonized lipase, wherein the modification and cross-linking process includes: performing a modification reaction on the immobilized bio-enzyme and a modifier, performing a cross-linking reaction on the obtained modified immobilized bio-enzyme and a cross-linking agent, washing and drying the obtained cross-linking reaction product, and the obtained product is an immobilized magnetic carbonized lipase. At this time, the lysine (ε-amino) produced by the enzyme can be covalently connected to the carrier as a linker molecule, and the enzyme is anchored to the carrier through the side chain of lysine (ε-amino) to enhance the activity, stability and effectiveness of enzyme fixation. At the same time, the lysine in the enzyme will react with carboxylic acid to form an additional hydrogen bond during the esterification process of astaxanthin and unsaturated fatty acids. This hydrogen bond will make the carboxylic acid molecule easier to leave, and the prepared lipase becomes an electrophilic catalyst, which makes the fatty acid and astaxanthin more capable of binding, and helps to accelerate the esterification reaction.

[0028] In the preparation process of the above-mentioned bio-enzyme, specific examples of the modifier include but are not limited to: at least one of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 3-aminopropyltriethoxysilane, N-bromosuccinimide and 4-ethyl-2,3-dioxy-1-piperazinecarbonyl chloride. The modifier is preferably used in the form of a solution, and the concentration of the modifier solution is preferably 0.01mol / L~10mol / L, such as 0.01, 0.1, 0.5, 1, 2, 4, 6, 8, 10mol / L or any value therebetween. The amount of the modifier solution is preferably 0.1%~10% of the total volume of the immobilized bio-enzyme, such as 0.1%, 1%, 2%, 4%, 6%, 8%, 10% or any value therebetween. The conditions of the modification reaction preferably include a temperature of 20°C to 70°C, such as 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C or any value therebetween; a time of 10min to 120min, such as 10min, 20min, 30min, 40min, 50min, 60min, 70min, 80min, 90min, 100min, 110min, 120min or any value therebetween. The cross-linking agent is preferably glutaraldehyde. Glutaraldehyde is cross-linked by forming a covalent bond between the enzyme molecule and the magnetic carbonized carrier molecule to form a magnetic immobilized lipase. The cross-linking agent is preferably used in the form of a solution, and the concentration of the cross-linking agent solution is preferably 0.1% to 50%, such as 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or any value therebetween. The amount of the cross-linking agent solution added is preferably 1% to 10% of the volume of the biological enzyme solution, such as 1%, 2%, 4%, 6%, 8%, 10% or any value therebetween. The conditions of the cross-linking reaction preferably include a temperature of 0°C to 20°C, such as 0°C, 2°C, 4°C, 6°C, 8°C, 10°C, 12°C, 14°C, 16°C, 18°C, 20°C or any value therebetween; and a time of 1h to 48h, such as 1h, 2h, 4h, 6h, 8h, 10h, 15h, 20h, 25h, 30h, 35h, 40h, 48h or any value therebetween. The washing liquid used for the washing is preferably a phosphate buffer solution. The number of washing times can be 2 to 3 times. The drying method is preferably freeze drying. The drying time is preferably 10 hours to 48 hours, such as 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, 22 hours, 25 hours, 28 hours, 30 hours, 32 hours, 35 hours, 38 hours, 40 hours, 42 hours, 45 hours, 48 ​​hours or any value therebetween.

[0029] The invention also provides the immobilized biological enzyme prepared by the method.

[0030] The method for synthesizing astaxanthin ester provided by the present invention comprises subjecting free astaxanthin to an esterification reaction with unsaturated fatty acid in the presence of the above-mentioned immobilized biological enzyme.

[0031] In the above-mentioned synthesis process of astaxanthin monoester, the free astaxanthin is extracted by fermentation. The purity of the free astaxanthin is preferably 40% to 100%, such as 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or any value therebetween.

[0032] In the above-mentioned synthesis process of astaxanthin monoester, specific examples of the unsaturated fatty acid include, but are not limited to, at least one of docosahexaenoic acid, arachidonic acid, oleic acid, linoleic acid, linolenic acid and palmitic acid.

[0033] In the synthesis process of the above-mentioned astaxanthin monoester, the molar ratio of free astaxanthin to unsaturated fatty acid is preferably 1:(0.1~10), such as 1:0.1, 1:0.5, 1:1, 1:2, 1:4, 1:6, 1:8, 1:10 or any value therebetween.

[0034] In the above-mentioned synthesis process of astaxanthin monoester, the dosage of the immobilized bioenzyme is preferably 0.01 to 10 times the weight of free astaxanthin, such as 0.01, 1, 2, 4, 6, 8, 10 times or any value therebetween.

[0035] In the above-mentioned synthesis process of astaxanthin monoester, the esterification reaction is preferably carried out in the presence of water. The addition of water can maintain the active conformation of the biological enzyme, which is more conducive to improving the yield of astaxanthin monoester. However, since the esterification reaction itself is a dehydration reaction, in order to make the reaction proceed smoothly, the amount of water added should not be too much. The amount of water added is preferably 0.01% to 1% of the total volume of the reaction system, such as 0.01%, 0.05%, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1% or any value therebetween.

[0036] In the above-mentioned synthesis process of astaxanthin monoester, the conditions of the esterification reaction preferably include a temperature of 10°C to 35°C, such as 10°C, 12°C, 15°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 35°C or any value therebetween; a stirring speed of 50r / min to 500r / min, such as 50, 100, 150, 200, 250, 300, 350, 400, 450, 500r / min or any value therebetween. During the esterification reaction, the reaction end point is determined by spotting the reaction liquid on the plate, and the reaction is terminated if there is a slight amount of astaxanthin diester generated. The purity of the synthetic product can be detected by HPLC high-performance liquid chromatography. The esterification reaction generally needs to be carried out in the presence of an organic solvent. The organic solvent can be selected from at least one of dichloromethane, chloroform, N,N-dimethylformamide, dimethyl sulfoxide, acetonitrile, acetone and ethyl acetate. In addition, the esterification reaction process should be protected from light and inert gas throughout.

[0037] The method for synthesizing astaxanthin ester provided by the present invention preferably further comprises washing the esterification reaction product with salt water and concentrating it, crystallizing the concentrate and then separating the solid and liquid, and the obtained filter residue is a free astaxanthin crystal product, and the free astaxanthin crystal product is dried and applied to the next batch of esterification reaction, and the obtained mother liquor is concentrated to dryness to obtain astaxanthin monoester. Wherein, the amount of the salt water is preferably 0.1 to 10 times the volume of the esterification reaction product, such as 0.1, 1, 2, 4, 6, 8, 10 times or any value therebetween. The salt water is a saturated solution of a monovalent salt such as potassium and sodium, and is preferably filtered through a 0.22 μm filter membrane when used. The conditions for concentrating the obtained mother liquor to dryness preferably include a temperature of 10°C to 35°C, such as 10°C, 15°C, 20°C, 25°C, 30°C, 35°C or any value therebetween; and a vacuum degree of 0.09 MPa or more, such as 0.09 MPa, 0.1 MPa, 0.2 MPa, 0.3 MPa, 0.4 MPa, 0.5 MPa, 0.6 MPa, 0.7 MPa, 0.8 MPa, 0.9 MPa, 1 MPa or any value therebetween.

[0038] In the present invention, the crystallization process preferably includes stirring and dissolving the concentrate in a crystallization solvent, wherein the crystallization solvent can dissolve astaxanthin monoester but basically cannot dissolve free astaxanthin, and cooling to a relatively low temperature for aging when a large amount of crystals are precipitated in the solution. Wherein, the crystallization solvent can be listed as at least one of petroleum ether, n-hexane, n-pentane, n-heptane, methanol, ethanol, isopropanol, acetone and ethyl acetate. The ratio of the crystallization solvent to the concentrate is preferably (1-10) mL: 1 g, such as 1 mL: 1 g, 2 mL: 1 g, 4 mL: 1 g, 6 mL: 1 g, 8 mL: 1 g, 10 mL: 1 g or any value therebetween. The dissolution conditions preferably include a temperature of 0°C to 40°C, such as 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C or any value therebetween; a time of 0.5h to 5h, such as 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h or any value therebetween. The aging conditions preferably include a temperature of 0°C to 10°C, such as 0°C, 2°C, 4°C, 6°C, 8°C, 10°C or any value therebetween; a time of 1h to 20h, such as 1h, 2h, 5h, 8h, 10h, 12h, 15h, 18h, 20h or any value therebetween.

[0039] The present invention will be described in detail below through examples.

[0040] In the following examples and comparative examples, the Antarctic Candida lipase liquids are all the same, and are all obtained by subjecting Pichia pastoris (CGMCC NO.10277) to slant seed culture, activated seed culture and primary seed culture, and then inoculating and fermenting in a fermenter containing BSM medium.

[0041] In the following examples and comparative examples, P123 (polyethylene oxide-polypropylene oxide-polyethylene oxide) was purchased from Qianyan Chemical Technology (Wuhan) Co., Ltd. with the brand name 106392-12-5; F127 (polyethylene oxide-polypropylene oxide-polyethylene oxide) was purchased from Sigma with the brand name 9003-11-6; SBS (copolymer of styrene-butadiene-3-chloropropylene) was purchased from Sigma with the brand name 9003-55-8.

[0042] Preparation Example 1 S1. Preparation of Derivatized Magnetic Carbon Support: Weigh 100g P123 (polyethylene oxide-polypropylene oxide-polyethylene oxide) and 200g potassium phosphate, mix and add to 1200mL of 1mol / L hydrochloric acid solution, stir and dissolve at 35°C, add 65g tetraethoxysilane after the polymer is dissolved, stir and react for 1h at the same temperature and store statically for 24h, age the formed gel in a high-pressure reactor at a temperature of 150°C and a pressure of 1MPa for 24h, filter to obtain solid particles, wash the solid particles with deionized water 3 times and dry them in a 110°C oven for 6h to obtain 268g of silicate solid particles. The silicate solid particles were added to 268mL of 0.3mol / L ferrous sulfate solution and soaked for 10h, and then the magnetically attracted solid particles were filtered out using a vacuum filtration device. The morphology and structure of the solid particles were characterized by scanning electron microscopy, and the results are as follows: Figure 1 As shown. Figure 1 It can be seen that the microporous structure of the carbon carrier particles after being soaked in ferrous sulfate solution remains intact and orderly. The solid particles were heated and carbonized in a muffle furnace at a temperature of 600°C for 2 hours to obtain 223g of a derivative magnetic carbon carrier with an ordered macroporous structure. The morphology and structure of the derivative magnetic carbon carrier were characterized by scanning electron microscopy. The results are shown in Figure 2. Figure 2 As shown. Figure 2 It can be seen that the structure of the derived magnetic carbon carrier is ordered.

[0043] S2. Immobilization of Candida lipase: 1L of Antarctic Candida lipase liquid was measured and separated into solid and liquid by a butterfly centrifuge to obtain 480g of lipase bacterial sludge. 1 times of pure water was added to the bacterial sludge, stirred evenly, and then centrifuged twice to obtain a fluidized bacterial sludge. The bacterial sludge was subjected to high-pressure homogenization and wall breaking at a pressure of 1200 bar to obtain 430mL of broken liquid, which was the biological enzyme liquid.

[0044] Dissolve 223g of the derived magnetic carbon carrier particles in 1100mL of 0.1mol / L potassium phosphate buffer and adjust the pH value to 7. Turn on the ultrasonic machine and ultrasonicate at 50℃ for 1min. Add 330mL of the biological enzyme solution under ultrasonic conditions and heat and mix at 70℃ for 1h. Continue to turn on the ultrasonication, add 17mL of 0.05mol / L 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride solution and stir for 10min. Then add 14.5mL of 15% glutaraldehyde solution. After stirring evenly, lower the temperature to 5℃ and stir for 24h to form gel particles. Filter the gel particles and wash them twice with 0.1mol / L potassium phosphate buffer solution. Then put them in a freeze dryer and dry them for 24h to obtain immobilized magnetic carbonized lipase. The scanning electron microscope image of the immobilized magnetic carbonized lipase is shown in Figure 3 and Figure 4 .from Figure 3 and Figure 4 It can be seen that after the enzyme was modified and immobilized on the carrier, the carrier structure did not change significantly, and the particles were uniform and the structure was compact.

[0045] Preparation Example 2 S1. Preparation of Derivatized Magnetic Carbon Support: Weigh 100g F127 (polyoxyethylene-polyoxypropylene-polyoxyethylene) and 1000g potassium phosphate, mix and add to 1200mL hydrochloric acid solution with a concentration of 1mol / L, stir and dissolve at 50℃, add 40g tetraethylsilane after the polymer is dissolved, stir and react for 1h at the same temperature and then store statically for 5h, age the formed gel in a high-pressure reactor at a temperature of 160℃ and a pressure of 1.2MPa for 10h, filter to obtain solid particles, wash the solid particles with deionized water 3 times and put them in a 100℃ oven to dry for 24h to obtain 255g silicate solid particles. The silicate solid particles are added to 500mL of ferrous sulfate solution with a concentration of 1mol / L and soaked for 24h, and then the magnetically attracted solid particles are filtered out by vacuum filtration equipment. The solid particles are heated and carbonized in a muffle furnace at a temperature of 500℃ for 10h to obtain 198g of a derivative magnetic carbon carrier with an ordered macroporous structure.

[0046] S2. Immobilization of Candida antarctica lipase: 1L of Antarctic Candida lipase liquid was measured and separated into solid and liquid by a butterfly centrifuge to obtain 465g of lipase bacterial sludge. 1 times of pure water was added to the bacterial sludge, stirred evenly, and then centrifuged twice to obtain a fluidized bacterial sludge. The bacterial sludge was subjected to high-pressure homogenization and wall breaking at a pressure of 500 bar to obtain 415mL of broken liquid, which was the biological enzyme liquid.

[0047] After dissolving 198 g of the derived magnetic carbon carrier particles in 990 mL of 0.1 mol / L potassium phosphate buffer, the pH value was adjusted to 5, and the ultrasonic machine was turned on to sonicate at 35°C for 10 minutes. Under ultrasonic conditions, 290 mL of the biological enzyme solution was added and heated and mixed at 35°C for 24 hours. The ultrasound was continued to be turned on, and 15 mL of 10 mol / L 3-aminopropyltriethoxysilane solution was added and stirred for 30 minutes. After that, 6.5 mL of 30% glutaraldehyde solution were added respectively. After stirring evenly, the temperature was lowered to 20°C and stirred for 1 hour to form gel particles. The gel particles were filtered out and washed twice with 0.1 mol / L potassium phosphate buffer solution, and then placed in a freeze dryer and dried for 10 hours to obtain immobilized magnetic carbonized lipase.

[0048] Preparation Example 3 S1. Preparation of Derivatized Magnetic Carbon Support: Weigh 100g SBS (styrene-butadiene-3-chloropropylene copolymer) and 500g potassium phosphate, mix and add to 1200mL hydrochloric acid solution with a concentration of 1mol / L, stir and dissolve at 45℃, add 80g diethoxydimethylsilane after the polymer is dissolved, stir and react for 1h at the same temperature and then store statically for 48h, age the formed gel in a high-pressure reactor at a temperature of 140℃ and a pressure of 0.8MPa for 48h and filter to obtain solid particles, wash the solid particles with deionized water 3 times and put them in a 105℃ oven for 10h to obtain 268g silicate solid particles. The silicate solid particles are added to 268mL of ferrous sulfate solution with a concentration of 0.5mol / L and soaked for 5h, and then the magnetically attracted solid particles are filtered out by vacuum filtration equipment. The solid particles are heated and carbonized in a muffle furnace at a temperature of 1000℃ for 10min to obtain 231g of a derivative magnetic carbon carrier with an ordered macroporous structure.

[0049] S2. Immobilization of Candida lipase: 1L of Antarctic Candida lipase liquid was measured and separated into solid and liquid by a butterfly centrifuge to obtain 520g of lipase bacterial sludge. 1 times of pure water was added to the bacterial sludge, stirred evenly, and then centrifuged twice to obtain a fluidized bacterial sludge. The bacterial sludge was subjected to high-pressure homogenization and wall breaking at a pressure of 1000 bar to obtain 505mL of broken liquid, which was the biological enzyme liquid.

[0050] After dissolving 231 g of the derived magnetic carbon carrier particles in 4460 mL of 0.1 mol / L potassium phosphate buffer, the pH value was adjusted to 8, and the ultrasonic machine was turned on to ultrasonicate for 60 min at 20 ° C. 450 mL of the biological enzyme solution was added under ultrasonic conditions and heated and mixed at 20 ° C for 40 h. The ultrasound was continued, and 17 mL of 5 mol / L 4-ethyl-2,3-dioxy-1-piperazinecarbonyl chloride solution was added and stirred for 120 min. Then 14.5 mL of 50% glutaraldehyde solution was added. After stirring evenly, the temperature was lowered to 0 ° C and stirred for 48 h to form gel particles. The gel particles were filtered out and washed twice with 0.1 mol / L potassium phosphate buffer solution, and then placed in a freeze dryer for 48 h to obtain immobilized magnetic carbonized lipase.

[0051] Preparation Example 4 The immobilized magnetic carbonized lipase was prepared according to the method of Preparation Example 1, except that the modification and cross-linking steps were not included. The specific steps are as follows: S1. Preparation of a derived magnetic carbon carrier: The same as Preparation Example 1, to obtain a derived magnetic carbon carrier.

[0052] S2. Immobilization of Candida lipase: Prepare a biological enzyme solution according to the method of Preparation Example 1. Dissolve 223g of the derived magnetic carbon carrier particles in 1100mL of 0.1mol / L potassium phosphate buffer and adjust the pH value to 7. Turn on the ultrasonic machine and ultrasonicate at 50°C for 1min. Add 330mL of the biological enzyme solution under ultrasonic conditions and heat and mix at 70°C for 1h. Filter and wash the obtained solid particles twice with 0.1mol / L potassium phosphate buffer solution, and then put them into a freeze dryer for drying for 24h to obtain immobilized magnetic carbonized lipase.

[0053] Comparative Preparation Example 1 The biological enzyme was prepared according to the method of Preparation Example 4, except that the steps of fixation, modification and cross-linking were not included. The specific steps were as follows: 1L of Antarctic Candida lipase liquid was measured and separated into solid and liquid by a butterfly centrifuge to obtain 480g of lipase bacterial sludge. 1 times of pure water was added to the bacterial sludge, stirred evenly, and then centrifuged twice to obtain a fluidized bacterial sludge. The bacterial sludge was subjected to high-pressure homogenization and wall breaking at a pressure of 1200 bar to obtain 430mL of broken liquid, which was the Antarctic Candida biological enzyme liquid.

[0054] Example 1 (Preparation of astaxanthin monoester by esterification synthesis using the immobilized magnetic carbonized lipase obtained in Preparation Example 1) Weigh 10g of free astaxanthin crystals, add 2.4g of oleic acid in a molar ratio of 1:0.5 and dissolve in 500mL of dichloromethane. After the free astaxanthin crystals are completely dissolved, add 1g of the immobilized magnetic carbonized lipase obtained in Preparation Example 1 and 0.05mL of water, stir and react at a rate of 500r / min at 25°C, operate in the dark and protect with nitrogen throughout the process. The reaction process is judged by the dot plate. If there is astaxanthin diester point color, the reaction is terminated immediately to obtain 496mL of reaction solution. Add 2 times the volume of saturated sodium chloride solution to the reaction solution, wash for 1h, and let it stand for stratification. When the oil layer is transparent, separate the lower organic phase and concentrate it under reduced pressure at 30°C to obtain 12.1g of concentrate.

[0055] Slowly drop 90mL of n-hexane into the concentrate, stir at 20°C for 2h, at which time a large amount of crystals precipitate in the solution, lower the temperature to 4°C and continue stirring for 2h, stop stirring, age for 10h, filter, and dry the crystals obtained by filtration in a 30°C vacuum oven. After drying, the crystals are 0.5g of free astaxanthin crystals, which are used in the next batch; concentrate the mother liquor obtained by filtration under a vacuum condition of 0.09MPa, and the concentrate is astaxanthin monoester. The weight, esterification rate and monoester yield of the final astaxanthin monoester are shown in Table 1.

[0056] Example 2 (Preparation of astaxanthin monoester by esterification synthesis using the immobilized magnetic carbonized lipase obtained in Preparation Example 2) Weigh 10g of free astaxanthin crystals, add 2.4g of oleic acid in a molar ratio of 1:0.5 and dissolve in 500mL of dichloromethane. After the free astaxanthin crystals are completely dissolved, add 5g of the immobilized magnetic carbonized lipase obtained in Preparation Example 2 and 0.1mL of water, stir the reaction at a rate of 50r / min at 25°C, operate in dark and nitrogen protection throughout the process. The reaction process is judged by the dot plate. If there is astaxanthin diester point color, the reaction is terminated immediately to obtain 497mL of reaction solution. Add 10 times the volume of saturated potassium chloride solution to the reaction solution, wash for 1h, and let it stand for stratification. When the oil layer is transparent, separate the lower organic phase and concentrate it under reduced pressure at 30°C to obtain 12.33g of concentrate.

[0057] Slowly drop 12mL of petroleum ether into the concentrate, stir at 40°C for 5h, at which time a large amount of crystals precipitate in the solution, lower the temperature to 10°C, continue stirring for 2h, stop stirring, age for 18h, filter, and dry the crystals obtained by filtration in a 30°C vacuum oven. After drying, the crystals are 0.35g of free astaxanthin crystals, which are used in the next batch; concentrate the mother liquor obtained by filtration under a vacuum condition of 0.09MPa, and the concentrate is astaxanthin monoester. The weight, esterification rate and monoester yield of the final astaxanthin monoester are shown in Table 1.

[0058] Example 3 (Using the immobilized magnetic carbonized lipase obtained in Preparation Example 1, linoleic acid and free astaxanthin crystals were used to perform esterification synthesis to prepare astaxanthin monoester) Weigh 10g of free astaxanthin crystals, add 2.35g of linoleic acid in a molar ratio of 1:0.5 and dissolve in 500mL of dichloromethane. After the free astaxanthin crystals are completely dissolved, add 10g of the immobilized magnetic carbonized lipase obtained in Preparation Example 1 and 5mL of water, stir and react at a rate of 200r / min at 25°C, operate in dark and nitrogen protection throughout the process. The reaction process is judged by the dot plate. If there is astaxanthin diester point color, the reaction is terminated immediately to obtain 493mL of reaction solution. Add 0.5 times the volume of saturated sodium chloride solution to the reaction solution, wash for 1h, and let stand for stratification. When the oil layer is transparent, separate the lower organic phase and concentrate it under reduced pressure at 30°C to obtain 11.98g of concentrate.

[0059] Slowly drop 12mL of isopropanol into the concentrate, stir for 5h at 5℃, at which time a large amount of crystals precipitate in the solution, lower the temperature to 4℃ and continue stirring for 2h, stop stirring, age for 1h, filter, and dry the crystals obtained by filtration in a 30℃ vacuum oven. After drying, the crystals are 0.58g of free astaxanthin crystals, which are used in the next batch; concentrate the mother liquor obtained by filtration under a vacuum condition of 0.09MPa, and the concentrate is astaxanthin monoester. The weight, esterification rate and monoester yield of the final astaxanthin monoester are shown in Table 1.

[0060] Example 4 Astaxanthin monoester was synthesized and prepared according to the method of Example 1, except that the immobilized magnetic carbonized lipase obtained in Preparation Example 1 was replaced by the immobilized magnetic carbonized lipase obtained in Preparation Example 3 in the same weight portion, and the other conditions were the same as in Example 1 to obtain astaxanthin monoester. The weight, esterification rate and monoester yield of the finally obtained astaxanthin monoester are shown in Table 1.

[0061] Example 5 Astaxanthin monoester was synthesized and prepared according to the method of Example 1, except that the immobilized magnetic carbonized lipase obtained in Preparation Example 1 was replaced by the immobilized magnetic carbonized lipase obtained in Preparation Example 4 in the same weight portion, and the other conditions were the same as in Example 1 to obtain astaxanthin monoester. The weight, esterification rate and monoester yield of the finally obtained astaxanthin monoester are shown in Table 1.

[0062] Example 6 Astaxanthin monoester was synthesized and prepared according to the method of Example 1, except that 0.05 mL of water was not added during the esterification reaction, and the other conditions were the same as in Example 1 to obtain astaxanthin monoester. The weight, esterification rate and monoester yield of the final astaxanthin monoester are shown in Table 1.

[0063] Comparative Example 1 Astaxanthin monoester was synthesized and prepared according to the method of Example 1, except that the immobilized magnetic carbonized lipase obtained in Preparation Example 1 was replaced by the same weight of Novozym435 lipase, and the other conditions were the same as in Example 1 to obtain astaxanthin monoester. The weight, esterification rate and monoester yield of the finally obtained astaxanthin monoester are shown in Table 1.

[0064] Comparative Example 2 Astaxanthin monoester was synthesized and prepared according to the method of Example 1, except that the immobilized magnetic carbonized lipase obtained in Preparation Example 1 was replaced by the same weight portion of the Antarctic Candida bioenzyme solution obtained in Comparative Preparation Example 1, and the other conditions were the same as in Example 1 to obtain astaxanthin monoester. The weight, esterification rate and monoester yield of the finally obtained astaxanthin monoester are shown in Table 1.

[0065] Table 1

[0066] It can be seen from the results in Table 1 that the immobilized bioenzyme obtained by the method provided by the present invention can catalyze free astaxanthin to efficiently synthesize astaxanthin monoester, and the yield of astaxanthin monoester is relatively high.

[0067] From the comparison between Example 1 and Example 5, it can be seen that when the method for preparing the immobilized bio-enzyme further includes the steps of modifying and cross-linking the immobilized bio-enzyme solution to obtain the immobilized magnetic carbonized lipase, the yield of astaxanthin monoester can be significantly improved.

[0068] From the comparison between Example 1 and Example 6, it can be seen that when the esterification reaction is carried out in the presence of water, it is more conducive to improving the yield of astaxanthin monoester.

[0069] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A method for preparing an immobilized biological enzyme, characterized in that: The method comprises the following steps: S1. Preparation of derived magnetic carbon carrier: an organic carrier, a phosphate and an organosilicon source are subjected to a sol-gel reaction in a hydrochloric acid solution, the obtained gel is subjected to high temperature aging to separate solid particles, the obtained solid particles are then washed with water and dried, and then the obtained silicate solid particles are immersed in an iron salt solution for solid-liquid separation, and then the obtained solid particles are pyrolyzed and carbonized to obtain a derived magnetic carbon carrier; S2. Enzyme immobilization: The derived magnetic carbon carrier is dispersed in a phosphate buffer solution, the pH value of the obtained dispersion is adjusted to 5-8 and then ultrasonically activated, and then the obtained activation solution and the biological enzyme solution are ultrasonically treated so that the biological enzyme solution is adsorbed on the carrier to obtain an immobilized biological enzyme.

2. The method for preparing an immobilized bioenzyme according to claim 1, characterized in that: In step S1, during the preparation of the derived magnetic carbon carrier, the sol-gel reaction method includes stirring and dissolving the organic carrier and phosphate in a hydrochloric acid solution, stirring and reacting the obtained solution with an organosilicon source, and then standing.

3. The method for preparing the immobilized bioenzyme according to claim 1, characterized in that: In step S1, the conditions in the preparation process of the derived magnetic carbon carrier include: The high temperature aging conditions include a temperature of 140°C to 160°C, a pressure of 0.8MPa to 1.2MPa, and a time of 10h to 48h; and / or, The drying conditions include a temperature of 100°C to 110°C and a time of 6h to 24h; and / or, The iron salt solution is a ferrous sulfate solution; and / or, The amount of the iron salt solution is 0.5 to 10 times the weight of the silicate solid particles; and / or, The soaking time is 1h to 24h; and / or, The pyrolysis carbonization conditions include a temperature of 500° C. to 1000° C. and a time of 10 min to 10 h.

4. The method for preparing an immobilized bioenzyme according to claim 1, characterized in that: In step S2, the biological enzyme solution is an antarctic Candida lipase solution.

5. The method for preparing an immobilized bioenzyme according to claim 1, characterized in that: In step S2, the enzyme immobilization conditions include: The ratio of the derivatized magnetic carbon carrier to the phosphate buffer solution is 1 g: (0.5-50) mL; and / or, The amount of the biological enzyme solution added is 0.1 to 1 times the volume of the phosphate buffer; and / or, The conditions of the ultrasonic activation include a temperature of 20°C to 50°C and a time of 1 min to 60 min; and / or, The ultrasonic treatment conditions include a temperature of 20° C. to 70° C. and a time of 1 h to 40 h.

6. The method for preparing an immobilized bioenzyme according to any one of claims 1 to 5, characterized in that: The method also includes the steps of modifying and cross-linking the immobilized bio-enzyme to obtain immobilized magnetic carbonized lipase. The modification and cross-linking process includes: subjecting the immobilized bio-enzyme to a modification reaction with a modifier, subjecting the obtained modified immobilized bio-enzyme to a cross-linking reaction with a cross-linking agent, washing and drying the obtained cross-linking reaction product, and the obtained product is the immobilized magnetic carbonized lipase.

7. The method for preparing an immobilized bioenzyme according to claim 6, characterized in that: The modification and cross-linking conditions include: The modifier is selected from at least one of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 3-aminopropyltriethoxysilane, N-bromosuccinimide and 4-ethyl-2,3-dioxy-1-piperazinecarbonyl chloride; and / or, The modifier is used in the form of a solution and the concentration of the modifier solution is 0.01 mol / L to 10 mol / L; and / or, The amount of the modifier solution is 0.1% to 10% of the total volume of the immobilized enzyme; and / or, The modification reaction conditions include a temperature of 20°C to 70°C and a time of 10 min to 120 min; and / or, The cross-linking agent is glutaraldehyde; and / or, The cross-linking agent is used in the form of a solution and the concentration of the cross-linking agent solution is 0.1% to 50%; and / or, The amount of the cross-linking agent solution added is 1% to 10% of the volume of the biological enzyme solution; and / or, The conditions of the cross-linking reaction include a temperature of 0°C to 20°C and a time of 1h to 48h; and / or, The washing solution used in the washing is a phosphate buffer solution; and / or, The drying method is freeze drying and the drying time is 10h~48h.

8. An immobilized biological enzyme prepared by the method according to any one of claims 1 to 7.

9. A method for synthesizing astaxanthin monoester, characterized in that: The method comprises subjecting free astaxanthin to an esterification reaction with unsaturated fatty acids in the presence of the immobilized biological enzyme according to claim 8.

10. The method for synthesizing astaxanthin monoester according to claim 9, characterized in that: The esterification reaction is carried out in the presence of water, and the amount of water added is 0.01% to 1% of the total volume of the reaction system.

11. The method for synthesizing astaxanthin monoester according to claim 9, characterized in that: The method also includes washing the esterification reaction product with brine and then concentrating it, crystallizing the concentrate and then separating the solid and liquid, and the obtained filter residue is a free astaxanthin crystal product. The free astaxanthin crystal product is dried and applied to the next batch of esterification reaction, and the obtained mother liquor is concentrated to dryness to obtain astaxanthin monoester.

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