An immobilized biocatalyst, a preparation method thereof, and a method for synthesizing astaxanthin monoester

Abstract

The present invention belongs to the field of astaxanthin synthesis, and particularly relates to an immobilized biocatalyst, a preparation method thereof, and a method for synthesizing astaxanthin monoester. The preparation method of the immobilized biocatalyst includes derivatizing a magnetic carbon support and immobilizing a biocatalyst solution on the derivatized magnetic carbon support. The immobilized biocatalyst obtained by the method provided by the present invention can efficiently catalyze the synthesis of astaxanthin monoester from free astaxanthin, and has a high yield of astaxanthin monoester. At the same time, it can effectively avoid magnetic loss caused by being removed from the material, solves the problem of difficult separation in the application of biocatalysts, and the obtained immobilized enzyme can be recycled.

Description

Technical Field

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

[0002] Astaxanthin (3,3′-dihydroxy-4,4-diketo-β,β′-carotene) is a xanthophyll carotenoid composed of eight isoprene units and belongs to keto-carotenoids. It widely exists in the biological world, especially in shrimps, crabs, fish of aquatic animals and feathers of birds. In addition, some algae can also produce astaxanthin, such as Haematococcus pluvialis which can carry out autotrophy and heterotrophy, and at the same time, Phaffia rhodozyma, Rhodotorula glutinis BF-6, Haematooccus pluvialis, etc. can also produce astaxanthin. The production of synthetic astaxanthin abroad includes Hoffmann-la roche AG in Switzerland and BASF in Germany.

[0003] Although astaxanthin is a carotenoid, its certain biological functions are far stronger than those of other carotenoids. Astaxanthin is a non-pro-vitamin carotenoid and cannot be converted into vitamin in animals, but it has extremely strong antioxidant properties, can scavenge free radicals in the body, regulate and reduce the damage caused by photochemistry, 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 antibodies related to cell-associated antigens in the body. In food, astaxanthin can not only color, but also effectively play the roles of freshness preservation, preventing color change, flavor change and deterioration. The anti-photosensitizing effect of astaxanthin is stronger than that of β-carotene. The pharmaceutical and food industries utilize the antioxidant, anti-inflammatory and immune-promoting effects of astaxanthin as drugs to prevent oxidative tissue damage and formulate health foods. Therefore, astaxanthin has broad application prospects.

[0004] In nature, astaxanthin exists alone or in a mixture in the form of free astaxanthin or astaxanthin fatty acid esters (monoester or diester), and 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, etc.

[0005] Astaxanthin prepared by microbial fermentation mainly exists in the free state. However, due to the presence of polyisoprene, free astaxanthin has hydrophobicity, which reduces its bioavailability and applicability. At the same time, because free astaxanthin has conjugated double bonds, carbonyl groups and hydroxyl groups, it has a high electron effect, which leads to its instability when exposed to air, high temperature and light, resulting in a significant reduction in its activity and affecting the use effect. Therefore, during feed processing and storage, pigments are easily degraded to a large extent. If astaxanthin can be converted into astaxanthin esters, it not only has the activity of astaxanthin, but also endows new functions due to the introduction of new groups, such as improved hydrophilicity and hydrophobicity, easy digestion and absorption, etc.

[0006] Currently, astaxanthin esters are mainly synthesized by chemical methods, using organic bases such as alkali metal alkoxides as catalysts and acyl chlorides as acylating reagents. The reaction process is complex, with high pollution. During the reaction process, astaxanthin is easily isomerized or degraded by heat, oxidation, etc., resulting in a reduction in the reaction yield. The separation and purification of the product are complex and the biological activity of the product is not high. Compared with chemical methods, the synthesis of astaxanthin esters by enzymatic catalysis has the advantages of mild reaction conditions, high catalytic efficiency, strong catalytic specificity, etc.

[0007] CN104513844A discloses a method for synthesizing astaxanthin succinate by coupling the catalysis, separation and reaction of lipase using Phaffia rhodozyma as the raw material. The raw material used in this method is the concentrate of the purified extraction solution. Yeast lipase and an acylating reagent (succinic anhydride) are directly added and catalytic esterification is carried out under certain conditions to synthesize astaxanthin succinate with better stability. This process uses a relatively low-purity extraction solution concentrate for esterification, which is likely 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, with a low recycling rate, resulting in high costs and being not conducive to large-scale production.

[0008] CN110218308A discloses a method for preparing astaxanthin methoxypolyethylene glycol acetate ester. Using chemically synthesized free astaxanthin or free astaxanthin from natural sources as the main skeleton, and methoxypolyethylene glycol acetate as the acyl donor and hydrophilic group, a catalytic synthesis reaction is carried out using a catalyst, and then astaxanthin methoxypolyethylene glycol acetate ester is obtained through purification. In this method, 4-dimethylaminopyridine (DMAP) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) are used as chemical catalysts, and the catalysts are removed by pickling, alkalinity, and brine washing in the later stage. However, the yield of astaxanthin ester prepared using chemical catalysts is relatively low, and there are safety problems caused by incomplete washing of chemical catalysts, as well as problems such as easy isomerization of astaxanthin ester during esterification, small production capacity, and high costs.

[0009] Furthermore, since astaxanthin is a secondary alcohol and there is a ketone group at the adjacent position occupying a relatively large spatial position, this makes the steric hindrance of astaxanthin in the esterification process larger, and it is difficult to catalytically esterify and synthesize astaxanthin esters using ordinary bioenzymes alone. In addition, free astaxanthin is prone to synthesize a mixture of astaxanthin monoesters and diesters during the esterification synthesis process. Since astaxanthin diesters have stronger lipophilicity than monoesters, astaxanthin diesters are not as easily digested and absorbed as astaxanthin monoesters. At the same time, the scavenging rates of astaxanthin monoesters for DPPH and ABTS are both better than those of astaxanthin diesters. Therefore, there is an urgent need in the art to develop a method that can efficiently catalytically esterify and synthesize astaxanthin esters, especially astaxanthin monoesters, to make the industrial production of astaxanthin monoesters from astaxanthin possible. Summary of the Invention

[0010] The first object of the present invention is to provide a method for preparing an immobilized bioenzyme, and the immobilized bioenzyme obtained by using this method can efficiently catalytically esterify and synthesize astaxanthin monoesters.

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

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

[0013] Specifically, the method for preparing the immobilized bioenzyme provided by the present invention includes the following steps:

[0014] S1. Preparation of a derivative magnetic carbon carrier: An organic carrier, phosphate, and organosilicon are subjected to a sol-gel reaction in a hydrochloric acid solution. The resulting gel is subjected to high-temperature aging and then the solid particles are separated. The obtained solid particles are washed with water and dried. Then, the obtained silicate solid particles are soaked in an iron salt solution and then the solid-liquid separation is carried out. Next, the obtained solid particles are pyrolytically carbonized to obtain a derivative magnetic carbon carrier;

[0015] S2. Enzyme immobilization: The derivative 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. Then, the obtained activated solution and the bioenzyme solution are ultrasonically treated so that the bioenzyme solution is adsorbed on the carrier to obtain an immobilized bioenzyme.

[0016] The method for synthesizing astaxanthin monoesters provided by the present invention includes subjecting free astaxanthin and an unsaturated fatty acid to an esterification reaction in the presence of the above immobilized bioenzyme.

[0017] The immobilized biocatalyst obtained by the method provided by the present invention can efficiently catalyze the synthesis of astaxanthin monoester from free astaxanthin, and the yield of astaxanthin monoester is relatively high. Moreover, the present invention magnetically modifies the carbon support and embeds magnetic species into the backbone of the support, which can effectively prepare a porous carbon support with stable magnetic modification. Compared with directly connecting magnetic nanoparticles to the surface of the material through weak interactions, magnetic loss caused by migration out of the material during use can be effectively avoided, and at the same time, the problems of limited stability and difficult separation in the application of biocatalysts are solved, and the obtained immobilized enzyme can be recycled. In addition, ultrasonic activation is introduced during the assimilation of enzyme immobilization in the present invention, which not only facilitates the firm connection between the support and the enzyme, but also prevents the aggregation of the support during the adsorption of biocatalyst, playing a dual role.

[0018] The principle of magnetic attraction of the derivative magnetic carbon support provided by the present invention is as follows: After impregnating silicate solid particles with iron salts and then pyrolytic carbonization, the Fe and Si elements in Fe-silicate on the obtained derivative magnetic carbon support exist in the forms of Fe 3 C and silicon sulfide, respectively. The etching effect of these substances on the porous carbon material framework during the formation process leads to changes in the support structure. Among them, the iron salt enters the pore structure of the silicate solid particles during the soaking process, and then undergoes high-temperature pyrolytic carbonization together with the silicate solid particles. Fe 2+ may be reduced by substances such as C in the framework of the porous carbon material and penetrate into the carbon framework to form Fe 3 C. The divalent S element in the iron salt (ferrous sulfate) is reduced and combined with Si 2+ to form silicon sulfide. Part of the O element forms volatile carbon oxides by combining with the C element in the framework and volatilizes. However, the Fe 3 C substance generated at this moment endows the prepared support with magnetism and enables it to be attracted by a magnet, and the generated magnetic effect can significantly enhance the later enzyme catalytic activity and improve the recovery rate and reuse rate after use.

[0019] In a preferred embodiment, the preparation method of the immobilized biocatalyst further includes the step of modifying and crosslinking the immobilized biocatalyst to obtain an immobilized magnetic carbonized lipase. The process of modification and crosslinking includes: performing a modification reaction on the immobilized biocatalyst and a modifier, performing a crosslinking reaction on the obtained modified immobilized biocatalyst and a crosslinking agent, washing the obtained crosslinking reaction product and then drying it, and the obtained product is the immobilized magnetic carbonized lipase. At this time, through the directional modification and immobilization of the amino acid residues constituting the enzyme catalytic active center, free astaxanthin can be efficiently catalyzed by the enzyme to esterify and synthesize astaxanthin monoester, and the obtained immobilized biocatalyst becomes insoluble in the reaction solution during the preparation process due to its immobilization and structural modification, and at the same time 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 astaxanthin tert-butyl alcohol with a large steric hindrance and unsaturated fatty acids to synthesize astaxanthin monoester. Description of the Drawings

[0020] Figure 1 Scanning electron micrograph (1 μm) of the solid particles obtained after magnetic attraction in Preparation Example 1;

[0021] Figure 2 Scanning electron micrograph (1 μm) of the derived magnetic carbon support obtained in Preparation Example 1;

[0022] Figure 3 Scanning electron micrograph (1 μm) of the immobilized magnetic carbonized lipase obtained in Preparation Example 1;

[0023] Figure 4 Scanning electron micrograph (100 μm) of the immobilized magnetic carbonized lipase obtained in Preparation Example 1. Detailed Description of the Invention

[0024] The method for preparing the immobilized biocatalyst provided by the present invention includes step S1 (i.e., the preparation of the derived magnetic carbon support) and step S2 (i.e., enzyme immobilization).

[0025] In the preparation process of the above-mentioned immobilized biocatalyst, step S1 specifically includes carrying out a sol-gel reaction on an organic carrier, a phosphate, and an organosilicon in a hydrochloric acid solution, subjecting the obtained gel to high-temperature aging and then separating out solid particles, washing the obtained solid particles with water and then drying, soaking the obtained solid particles of silicate in an iron salt solution and then separating the solid and liquid, and then subjecting the obtained solid particles to pyrolytic carbonization to obtain a derived magnetic carbon support.

[0026] In the preparation process of the above-mentioned immobilized biocatalyst, the derived magnetic carbon support, as a host material, not only has a high enzyme capacity but also has good stability and mechanical strength. The derived magnetic carbon support has characteristics such as a large specific surface area, a high porosity, and a stable structure, and is extremely suitable for enzyme immobilization. Specifically, in the present invention, mesoporous silicate solid particles are first prepared, and then through the improvement of pyrolytic carbonization magnetic attraction, not only can its porosity and pore area be increased, but also it can have super magnetism.

[0027] In the preparation process of the above-mentioned immobilized biocatalyst, since the structure of the carbon material mainly depends on covalent bonds, high-temperature pyrolytic carbonization can not only improve the chemical stability of the obtained carbon support, but also remove some surfactants on the surface of the carbon support particles to make the pores of the obtained carbon support particles larger. In the present invention, the synthesized silicate solid particles are impregnated with iron salts and then pyrolytically carbonized to prepare a macroporous derivative magnetic carbon support, which can increase the reuse rate of the enzyme. At the same time, a major limitation in the large-scale industrial application of immobilized enzymes is the high cost introduced by their separation and recovery. In this regard, the enzyme immobilization support of the present invention is magnetically modified, and magnetic species are embedded in the backbone of the support, so that a porous carbon material with stable magnetic modification can be effectively obtained. Compared with directly using magnetic nanoparticles through weak interactions, the obtained derivative magnetic carbon support in the present invention may avoid magnetic loss caused by being removed from the material during use, and at the same time solves the problems of stability limitation and separation difficulty in the application of immobilized lipase.

[0028] In the preparation process of the derivative magnetic carbon support mentioned in step S1 in the preparation process of the above-mentioned immobilized biocatalyst, the sol-gel reaction method includes stirring and dissolving an organic support and phosphate in a hydrochloric acid solution, and stirring and mixing the obtained solution with an organosilicon source and then standing. Among them, the mass ratio of the organic support 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 (K 2 PO 4). The phosphates added during the sol-gel process form particles with different morphologies during the static 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 to 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 between them. The amount of the hydrochloric acid solution is preferably 1 to 50 times the total weight of the organic carrier, phosphates, and organosilicon source, such as 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 times or any value between them. The temperature for 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 between them. The organic carrier is preferably a triblock copolymer, and specific examples include at least one of SBS (copolymer of styrene-butadiene-3-chloropropene), F127 (polyethylene oxide-polypropylene oxide-polyethylene oxide), 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 to 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 between them. The conditions for the stirring reaction 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 between them; and a time of 10 min to 5 h, such as 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h or any value between them. The conditions for the static state 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 between them; and a time of 1 h to 48 h, such as 1 h, 5 h, 10 h, 12 h, 24 h, 36 h, 40 h, 44 h, 48 h or any value between them.

[0029] In the preparation process of the immobilized biocatalyst mentioned above, during the preparation process of the derived magnetic carbon support mentioned in step S1, the conditions for high-temperature aging 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 0.8 MPa to 1.2 MPa, such as 0.8 MPa, 0.9 MPa, 1 MPa, 1.1 MPa, 1.2 MPa or any value therebetween; and a time of 10 h to 48 h, such as 10 h, 15 h, 20 h, 24 h, 30 h, 36 h, 40 h, 44 h, 48 h or any value therebetween. The conditions for drying 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; and a time of 6 h to 24 h, such as 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, 24 h or any value therebetween. The iron salt solution is particularly preferably a ferrous sulfate solution. The concentration of the iron salt solution is preferably 0.01 mol / L to 1 mol / 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, 1 mol / L or any value therebetween. The dosage 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 1 h to 24 h, such as 1 h, 2 h, 5 h, 8 h, 10 h, 12 h, 15 h, 18 h, 20 h, 24 h or any value therebetween. The conditions for pyrolytic carbonization preferably include a temperature of 500°C to 1000°C, such as 500°C, 600°C, 700°C, 800°C, 900°C, 1000°C or any value therebetween; and a time of 10 min to 10 h, such as 10 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h 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 of washing the solid particles with water is preferably 2 to 3 times. The solid-liquid separation can be vacuum filtration.

[0030] In the preparation process of the above-mentioned biological enzyme, in the enzyme immobilization process mentioned in step S2, the biological enzyme solution can be various existing enzyme solutions capable of catalyzing the esterification reaction of astaxanthin and unsaturated fatty acids to produce astaxanthin monoesters, and is particularly preferably Candida antarctica lipase solution. The source of the biological enzyme solution is not particularly limited, and it can be obtained through commercial purchase or prepared according to various existing methods. In a preferred embodiment, the biological enzyme solution is prepared by the following method: separating the Candida antarctica lipase solution by solid-liquid separation, then washing the obtained bacterial sludge with water and breaking the wall, and the obtained broken wall solution is the biological enzyme solution. Among them, the solid-liquid separation method only needs to be able to separate the liquid and the bacterial sludge in the Candida antarctica lipase solution, and can be selected from at least one of pressure filtration, suction filtration, centrifugal separation, and membrane filtration. The purpose of the water washing is to remove the salts on the surface of the bacterial sludge. The number of water washing times can be reasonably selected according to the actual situation, generally 1 to 5 times. The purpose of breaking the wall is to fully release the target substances inside the cells and enhance its enzyme activity. The wall breaking is preferably carried out in a high-pressure homogenizer. Among them, the pressure during the operation of the high-pressure homogenizer is preferably controlled at 500 to 2000 bar, such as 500 bar, 800 bar, 1000 bar, 1200 bar, 1500 bar, 1800 bar, 2000 bar or any value between them.

[0031] In the preparation process of the above-mentioned bio-enzyme, in the enzyme immobilization process mentioned in step S2, the concentration of the phosphate buffer solution is preferably 0.01 mol / L to 10 mol / L, such as 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mol / L or any value therebetween. The dosage ratio of the derived magnetic carbon carrier to the phosphate buffer solution is preferably 1 g:(0.5 - 50) mL. Specifically, based on the dosage of the derived magnetic carbon carrier being 1 g, the dosage of the phosphate buffer solution can be 0.5, 1, 2, 5, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50 mL. The conditions of 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; and 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 addition amount of the bio-enzyme solution is preferably 0.1 to 1 times the volume of the phosphate buffer solution, such as 0.1, 0.2, 0.4, 0.6, 0.8, 1 times or any value therebetween. The conditions of 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 1 h to 40 h, such as 1 h, 5 h, 10 h, 15 h, 20 h, 25 h, 30 h, 35 h, 40 h, 45 h, 50 h or any value therebetween.

[0032] 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. The process of modification and cross-linking 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 the immobilized magnetic carbonized lipase. At this time, the lysine (ε-amino group) produced by the enzyme can be used as a linker molecule to be covalently connected to the carrier, and the enzyme is anchored to the carrier through the side chain of lysine (ε-amino group) so as to enhance the activity, stability and effectiveness of enzyme immobilization. At the same time, lysine in the enzyme will react with carboxylic acid during the esterification process of astaxanthin and unsaturated fatty acid to form additional hydrogen bonds. This hydrogen bond will make the carboxylic acid molecule more likely to leave, and the prepared lipase becomes an electrophilic catalyst, making the binding ability of fatty acid and astaxanthin stronger, which helps to accelerate the esterification reaction.

[0033] 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-dioxo-1-piperazinecarboxylic acid chloride. The modifier is preferably used in the form of a solution, and the concentration of the modifier solution is preferably 0.01 mol / L to 10 mol / L, such as 0.01, 0.1, 0.5, 1, 2, 4, 6, 8, 10 mol / L or any value therebetween. The dosage of the modifier solution is preferably 0.1% to 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; and a time of 10 min to 120 min, such as 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min, 120 min or any value therebetween. The cross-linking agent is preferably glutaraldehyde. Glutaraldehyde forms a covalent bond between the enzyme molecule and the magnetic carbonized carrier molecule for cross-linking, thereby forming 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 addition amount of the cross-linking agent solution is preferably 1% to 10% of the volume of the bio-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 1 h to 48 h, such as 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 15 h, 20 h, 25 h, 30 h, 35 h, 40 h, 48 h or any value therebetween. The washing liquid used for 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 h to 48 h, such as 10 h, 12 h, 15 h, 18 h, 20 h, 22 h, 25 h, 28 h, 30 h, 32 h, 35 h, 38 h, 40 h, 42 h, 45 h, 48 h or any value therebetween.

[0034] The present invention also provides an immobilized bio-enzyme prepared by the above method.

[0035] The synthesis method of astaxanthin ester provided by the present invention includes carrying out an esterification reaction between free astaxanthin and unsaturated fatty acid in the presence of the above immobilized biocatalyst.

[0036] In the synthesis process of the above astaxanthin monoester, the free astaxanthin is obtained by extraction through fermentation method. Among them, the purity of the free astaxanthin is preferably 40% - 100%, such as 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or any value between them.

[0037] In the synthesis process of the above 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.

[0038] In the synthesis process of the above astaxanthin monoester, the molar ratio of the free astaxanthin to the 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 between them.

[0039] In the synthesis process of the above astaxanthin monoester, the dosage of the immobilized biocatalyst is preferably 0.01 - 10 times the weight of the free astaxanthin, such as 0.01, 1, 2, 4, 6, 8, 10 times or any value between them.

[0040] In the synthesis process of the above 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 biocatalyst, 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 addition amount of water should not be too much. The addition amount of water is preferably 0.01% - 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 between them.

[0041] In the synthesis process of the above-mentioned 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 between them; the stirring speed is 50 r / min to 500 r / min, such as 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 r / min or any value between them. During the esterification reaction, the end point of the reaction is judged by spotting the reaction solution on a TLC plate. If a small amount of astaxanthin diester is formed, the reaction is terminated. The purity of the synthesis 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 kept away from light throughout and protected by an inert gas.

[0042] The synthesis method of astaxanthin ester provided by the present invention preferably further includes washing the esterification reaction product with brine and then concentrating it, crystallizing the concentrate and then performing solid-liquid separation. The obtained filter residue is the free astaxanthin crystal product. After drying, the free astaxanthin crystal product is applied to the next batch of esterification reaction, and the mother liquor is concentrated to dryness to obtain astaxanthin monoester. Among them, the dosage of the brine 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 between them. The brine is a saturated solution of monovalent salts such as potassium and sodium, and it 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 between them; the vacuum degree is above 0.09 MPa, 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 between them.

[0043] In the present invention, the crystallization process preferably includes stirring and dissolving the concentrate in a crystallization solvent that can dissolve astaxanthin monoester but can hardly dissolve free astaxanthin, and cooling to a lower temperature for aging when a large amount of crystals precipitate in the solution. Among them, the crystallization solvent may include 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; and a time of 0.5 h to 5 h, such as 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 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; and a time of 1 h to 20 h, such as 1 h, 2 h, 5 h, 8 h, 10 h, 12 h, 15 h, 18 h, 20 h, or any value therebetween.

[0044] The present invention will be described in detail below through examples.

[0045] In the following examples and comparative examples, the Candida antarctica lipase solution is the same, and is obtained by performing slant seed culture, activated seed culture, and primary seed culture on Pichia pastoris (CGMCC NO. 10277), and then inoculating it into a fermentation tank containing BSM medium for fermentation culture.

[0046] In the following examples and comparative examples, P123 (polyethylene oxide-polypropylene oxide-polyethylene oxide) is purchased from Qianyan Chemical Technology (Wuhan) Co., Ltd., with the product number 106392-12-5; F127 (polyoxyethylene-polyoxypropylene-polyoxyethylene) is purchased from Sigma Company, with the product number 9003-11-6; SBS (copolymer of styrene-butadiene-3-chloropropene) is purchased from Sigma Company, with the product number 9003-55-8.

[0047] Preparation Example 1

[0048] S1. Preparation of a derivative magnetic carbon carrier:

[0049] Weigh 100 g of P123 (polyethylene oxide - polypropylene oxide - polyethylene oxide) and 200 g of potassium phosphate, mix them, and add them to 1200 mL of a hydrochloric acid solution with a concentration of 1 mol / L. Stir and dissolve at 35 °C. After the polymer is dissolved, add 65 g of tetraethoxysilane. Stir and react for 1 h at the same temperature, then statically preserve for 24 h. Age the formed gel in a high-pressure reactor at a temperature of 150 °C and a pressure of 1 MPa for 24 h, and then filter to obtain solid particles. Wash the solid particles 3 times with deionized water and then place them in an oven at 110 °C to dry for 6 h to obtain 268 g of silicate solid particles. Add the silicate solid particles to 268 mL of a ferrous sulfate solution with a concentration of 0.3 mol / L and soak for 10 h, and then filter out the magnetically attracted solid particles using a vacuum filtration device. The morphology and structure of the solid particles are characterized by scanning electron microscopy, and the results are as shown in Figure 1 shown. It can be seen from Figure 1 that the microporous structure in the carbon support particles remains intact and ordered after being soaked in the ferrous sulfate solution. Heat and carbonize the solid particles in a muffle furnace at 600 °C for 2 h to obtain 223 g of a derived magnetic carbon support with an ordered macroporous structure. The morphology and structure of the derived magnetic carbon support are characterized by scanning electron microscopy, and the results are as shown in Figure 2 shown. It can be seen from Figure 2 that the structure of the derived magnetic carbon support is ordered.

[0050] S2. Immobilization of Candida antarctica lipase:

[0051] Measure 1 L of Candida antarctica lipase solution, perform solid-liquid separation with a disc centrifuge to obtain 480 g of lipase bacterial sludge. Add 1 times pure water to the bacterial sludge, stir evenly, and then perform secondary centrifugation to obtain a bacterial sludge with a flowing state. Subject the bacterial sludge to high-pressure homogenization and cell wall breaking at a pressure of 1200 bar. After cell wall breaking, 430 mL of a cell wall-breaking solution is obtained, which is a biological enzyme solution.

[0052] Dissolve 223 g of derived magnetic carbon support particles in 1100 mL of a potassium phosphate buffer solution with a concentration of 0.1 mol / L, then adjust the pH value to 7. Turn on the ultrasonic machine and ultrasonicate for 1 min at 50 °C. Under ultrasonic conditions, add 330 mL of the biological enzyme solution and heat and mix at 70 °C for 1 h. Continue to turn on the ultrasonic, add 17 mL of a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution with a concentration of 0.05 mol / L, stir for 10 min, and then add 14.5 mL of a 15% glutaraldehyde solution. Stir evenly, then lower the temperature to 5 °C and stir and react for 24 h to form gel particles. Filter out the gel particles and wash them 2 times with a 0.1 mol / L potassium phosphate buffer solution, and then place them in a freeze dryer to dry for 24 h to obtain immobilized magnetic carbonized lipase. The scanning electron micrograph of the immobilized magnetic carbonized lipase is shown in Figure 3 andFigure 4 From Figure 3 and Figure 4 It can be seen that after the enzyme is modified and immobilized on the carrier, the structure of the carrier does not change significantly, and the particles are uniform and the structure is compact.

[0053] Preparation Example 2

[0054] S1. Preparation of the derivatized magnetic carbon carrier:

[0055] Weigh 100 g of F127 (polyoxyethylene - polyoxypropylene - polyoxyethylene) and 1000 g of potassium phosphate, mix them and add them to 1200 mL of a 1 mol / L hydrochloric acid solution. Stir and dissolve at 50 °C. After the polymer is dissolved, add 40 g of tetraethylsilane, stir and react at the same temperature for 1 h, then statically preserve for 5 h. Age the formed gel in a high-pressure reactor at a temperature of 160 °C and a pressure of 1.2 MPa for 10 h, then filter to obtain solid particles. Wash the solid particles 3 times with deionized water and then put them in an oven at 100 °C to dry for 24 h to obtain 255 g of silicate solid particles. Add the silicate solid particles to 500 mL of a 1 mol / L ferrous sulfate solution and soak for 24 h, then filter out the magnetically attracted solid particles using a vacuum filtration device. Heat and carbonize the solid particles in a muffle furnace at 500 °C for 10 h to obtain 198 g of a derivatized magnetic carbon carrier with an ordered macroporous structure.

[0056] S2. Immobilization of Candida antarctica lipase:

[0057] Measure 1 L of Candida antarctica lipase solution, perform solid-liquid separation with a disc centrifuge to obtain 465 g of lipase bacterial sludge. Add 1 times pure water to the bacterial sludge, stir evenly, and then perform secondary centrifugation to obtain a bacterial sludge with a flowing state. Subject the bacterial sludge to high-pressure homogenization and cell wall breaking at a pressure of 500 bar. After cell wall breaking, obtain 415 mL of a cell wall broken solution, which is a biological enzyme solution.

[0058] Dissolve 198 g of derivatized magnetic carbon carrier particles in 990 mL of a 0.1 mol / L potassium phosphate buffer solution, then adjust the pH value to 5. Turn on the ultrasonic machine and ultrasonicate for 10 min at 35 °C. Under ultrasonic conditions, add 290 mL of the biological enzyme solution and heat and mix at 35 °C for 24 h. Continue to turn on the ultrasonic, add 15 mL of a 10 mol / L 3-aminopropyltriethoxysilane solution, stir for 30 min, then add 6.5 mL of a 30% glutaraldehyde solution, stir evenly, then lower the temperature to 20 °C and stir and react for 1 h to form gel particles. Filter out the gel particles, wash them 2 times with a 0.1 mol / L potassium phosphate buffer solution, and then put them in a freeze dryer to dry for 10 h to obtain immobilized magnetic carbonized lipase.

[0059] Preparation Example 3

[0060] S1. Preparation of Derived Magnetic Carbon Carrier:

[0061] Weigh 100 g of SBS (copolymer of styrene-butadiene-3-chloropropene) and 500 g of potassium phosphate, mix them and add them into 1200 mL of hydrochloric acid solution with a concentration of 1 mol / L. Stir and dissolve at 45 °C. After the polymer is dissolved, add 80 g of diethoxydimethylsilane, stir and react at the same temperature for 1 h, then statically preserve for 48 h. Age the formed gel in a high-pressure reactor at a temperature of 140 °C and a pressure of 0.8 MPa for 48 h, then filter to obtain solid particles. Wash the solid particles 3 times with deionized water and put them in an oven at 105 °C to dry for 10 h to obtain 268 g of silicate solid particles. Add the silicate solid particles into 268 mL of ferrous sulfate solution with a concentration of 0.5 mol / L and soak for 5 h, then filter out the magnetically attracted solid particles using a vacuum filtration device. Heat and carbonize the solid particles in a muffle furnace at 1000 °C for 10 min to obtain 231 g of derived magnetic carbon carrier with an ordered macroporous structure.

[0062] S2. Immobilization of Candida Lipase:

[0063] Measure 1 L of Candida antarctica lipase solution, perform solid-liquid separation with a disc centrifuge to obtain 520 g of lipase bacterial sludge. Add 1 time of pure water to the bacterial sludge, stir evenly, and then perform secondary centrifugation to obtain a bacterial sludge with a flowing state. Subject the bacterial sludge to high-pressure homogenization and cell wall breaking at a pressure of 1000 bar. After cell wall breaking, obtain 505 mL of broken cell solution, which is a biological enzyme solution.

[0064] Dissolve 231 g of derived magnetic carbon carrier particles in 4460 mL of potassium phosphate buffer solution with a concentration of 0.1 mol / L, then adjust the pH value to 8. Turn on the ultrasonic machine and ultrasonicate at 20 °C for 60 min. Under ultrasonic conditions, add 450 mL of biological enzyme solution and heat and mix at 20 °C for 40 h. Continue to turn on the ultrasonic, add 17 mL of 4-ethyl-2,3-dioxo-1-piperazinecarbonyl chloride solution with a concentration of 5 mol / L, stir for 120 min, then add 14.5 mL of glutaraldehyde solution with a concentration of 50%, stir evenly, then lower the temperature to 0 °C and stir and react for 48 h to form gel particles. Filter out the gel particles and wash them 2 times with 0.1 mol / L potassium phosphate buffer solution, then put them in a freeze dryer and dry for 48 h to obtain immobilized magnetic carbonized lipase.

[0065] Preparation Example 4

[0066] Prepare immobilized magnetic carbonized lipase according to the method of Preparation Example 1, except that the steps of modification and cross-linking are not included. The specific steps are as follows:

[0067] S1. Preparation of the derived magnetic carbon carrier: Follow Preparation Example 1 to obtain the derived magnetic carbon carrier.

[0068] S2. Immobilization of Candida lipase: Prepare the bioenzyme solution according to the method of Preparation Example 1. Dissolve 223 g of the derived magnetic carbon carrier particles in 1100 mL of a potassium phosphate buffer solution with a concentration of 0.1 mol / L, then adjust the pH value to 7. Turn on the ultrasonic machine and ultrasonicate for 1 min at 50 °C. Under ultrasonic conditions, add 330 mL of the bioenzyme solution and heat and mix at 70 °C for 1 h. Filter, wash the obtained solid particles twice with a 0.1 mol / L potassium phosphate buffer solution, and then place them in a freeze dryer to dry for 24 h to obtain immobilized magnetic carbonized lipase.

[0069] Comparative Preparation Example 1

[0070] Prepare the bioenzyme according to the method of Preparation Example 4, except that the steps of immobilization, modification, and crosslinking are not included. The specific steps are as follows:

[0071] Measure 1 L of Candida antarctica lipase solution, perform solid-liquid separation with a disc centrifuge to obtain 480 g of lipase bacterial sludge. Add 1 times pure water to the bacterial sludge, stir evenly, and then perform secondary centrifugation to obtain a bacterial sludge with a flowing state. Subject this bacterial sludge to high-pressure homogenization and cell wall breaking at a pressure of 1200 bar. After cell wall breaking, 430 mL of broken cell solution is obtained, which is the Candida antarctica bioenzyme solution.

[0072] Example 1 (Preparation of astaxanthin monoester by esterification synthesis using the immobilized magnetic carbonized lipase obtained in Preparation Example 1)

[0073] Weigh 10 g of free astaxanthin crystalline product, add 2.4 g of oleic acid according to a molar ratio of 1:0.5, dissolve in 500 mL of dichloromethane. After the free astaxanthin crystalline product is completely dissolved, add 1 g of the immobilized magnetic carbonized lipase obtained in Preparation Example 1 and 0.05 mL of water, and stir and react at a rate of 500 r / min at 25 °C. Operate under light protection throughout the process and carry out nitrogen protection. The reaction process is judged by thin-layer chromatography. If the astaxanthin diester spot shows color, immediately terminate the reaction to obtain 496 mL of the reaction solution. Add 2 times the volume of saturated sodium chloride solution to the reaction solution, wash for 1 h, let it stand for stratification. When the oil layer is clear, separate the lower organic phase and concentrate it to dryness under reduced pressure at 30 °C to obtain 12.1 g of the concentrate.

[0074] Slowly add 90 mL of n-hexane to the concentrate, stir at 20 °C for 2 h. At this time, a large amount of crystals precipitate in the solution. Lower the temperature to 4 °C and continue stirring for 2 h, then stop stirring. Age for 10 h and filter. Put the filtered crystalline product into a vacuum oven at 30 °C for drying. After drying, the crystalline product is 0.5 g of free astaxanthin crystalline product, which is used for the next batch; Concentrate the filtered mother liquor to dryness under a vacuum of 0.09 MPa. The concentrate is astaxanthin monoester. The weight, esterification rate, and monoester yield of the finally obtained astaxanthin monoester are shown in Table 1.

[0075] Example 2 (Using the immobilized magnetic carbonized lipase obtained in Preparation Example 2 for esterification synthesis to prepare astaxanthin monoester)

[0076] Weigh 10 g of free astaxanthin crystalline product, add 2.4 g of oleic acid according to a molar ratio of 1:0.5 and dissolve it in 500 mL of dichloromethane. After the free astaxanthin crystalline product is completely dissolved, add 5 g of the immobilized magnetic carbonized lipase obtained in Preparation Example 2 and 0.1 mL of water, and stir and react at 25 °C at a rate of 50 r / min. The whole process is carried out under light protection and nitrogen protection. The reaction process is judged by TLC. If the spot of astaxanthin diester shows color, immediately terminate the reaction to obtain 497 mL of reaction solution. Add 10 times the volume of saturated potassium chloride solution to the reaction solution, wash for 1 h, let it stand for layering. When the oil layer is clear, separate the lower organic phase and concentrate it to dryness under reduced pressure at 30 °C to obtain 12.33 g of concentrate.

[0077] Slowly add 12 mL of petroleum ether to the concentrate, stir at 40 °C for 5 h. At this time, a large amount of crystals precipitate in the solution. Lower the temperature to 10 °C and continue stirring for 2 h, then stop stirring. Age for 18 h and filter. Put the filtered crystalline product into a vacuum oven at 30 °C for drying. After drying, the crystalline product is 0.35 g of free astaxanthin crystalline product, which is used for the next batch; Concentrate the filtered mother liquor to dryness under a vacuum of 0.09 MPa. The concentrate is astaxanthin monoester. The weight, esterification rate, and monoester yield of the finally obtained astaxanthin monoester are shown in Table 1.

[0078] Example 3 (Using the immobilized magnetic carbonized lipase obtained in Preparation Example 1, using linoleic acid and free astaxanthin crystalline product for esterification synthesis to prepare astaxanthin monoester)

[0079] Weigh 10 g of free astaxanthin crystalline product, add 2.35 g of linoleic acid according to a molar ratio of 1:0.5, dissolve it in 500 mL of dichloromethane. After the free astaxanthin crystalline product is completely dissolved, add 10 g of the immobilized magnetic carbonized lipase obtained in Preparation Example 1 and 5 mL of water, and stir and react at a rate of 200 r / min at 25 °C. The whole process is carried out under light protection and nitrogen protection. The reaction process is judged by thin-layer chromatography. If the spot of astaxanthin diester shows color, immediately terminate the reaction to obtain 493 mL of the reaction solution. Add 0.5 times the volume of saturated sodium chloride solution to the reaction solution, wash for 1 h, let it stand for stratification. When the oil layer is clear, separate the lower organic phase and concentrate it to dryness under reduced pressure at 30 °C to obtain 11.98 g of the concentrate.

[0080] Slowly add 12 mL of isopropanol dropwise to the concentrate, stir at 5 °C for 5 h. At this time, a large amount of crystals precipitate in the solution. Lower the temperature to 4 °C, continue to stir for 2 h, stop stirring, age for 1 h, filter. Put the filtered crystalline product into a vacuum oven at 30 °C for drying. The dried crystalline product is 0.58 g of free astaxanthin crystalline product, which is used for the next batch; Concentrate the filtered mother liquor to dryness under a vacuum degree of 0.09 MPa. The concentrate is astaxanthin monoester. The weight, esterification rate and monoester yield of the finally obtained astaxanthin monoester are shown in Table 1.

[0081] Example 4

[0082] Synthesize and prepare astaxanthin monoester according to the method of Example 1. The difference is that the immobilized magnetic carbonized lipase obtained in Preparation Example 1 is replaced with the immobilized magnetic carbonized lipase obtained in Preparation Example 3 with the same weight, and the other conditions are the same as those 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.

[0083] Example 5

[0084] Synthesize and prepare astaxanthin monoester according to the method of Example 1. The difference is that the immobilized magnetic carbonized lipase obtained in Preparation Example 1 is replaced with the immobilized magnetic carbonized lipase obtained in Preparation Example 4 with the same weight, and the other conditions are the same as those 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.

[0085] Example 6

[0086] Synthesize and prepare astaxanthin monoester according to the method of Example 1. The difference is that 0.05 mL of water is not added during the esterification reaction, and the other conditions are the same as those 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.

[0087] Comparative Example 1

[0088] The astaxanthin monoester was synthesized and prepared according to the method of Example 1, except that the immobilized magnetic carbonized lipase obtained from Preparation Example 1 was replaced with Novozym 435 lipase in the same weight portion, and the other conditions were the same as those in Example 1, and the astaxanthin monoester was obtained. The weight, esterification rate and monoester yield of the finally obtained astaxanthin monoester are shown in Table 1.

[0089] Comparative Example 2

[0090] The astaxanthin monoester was synthesized and prepared according to the method of Example 1, except that the immobilized magnetic carbonized lipase obtained from Preparation Example 1 was replaced with the Candida antarctica bioenzyme solution obtained from Comparative Preparation Example 1 in the same weight portion, and the other conditions were the same as those in Example 1, and the astaxanthin monoester was obtained. The weight, esterification rate and monoester yield of the finally obtained astaxanthin monoester are shown in Table 1.

[0091] Table 1

[0092]

[0093] It can be seen from the results in Table 1 that the immobilized biocatalyst obtained by the method provided by the present invention can catalyze the efficient synthesis of astaxanthin monoester from free astaxanthin, and the yield of astaxanthin monoester is relatively high.

[0094] It can be seen from the comparison between Example 1 and Example 5 that when the preparation method of the immobilized biocatalyst further includes the step of modifying and crosslinking the immobilized biocatalyst solution to obtain the immobilized magnetic carbonized lipase, the yield of astaxanthin monoester can be significantly improved.

[0095] It can be seen from the comparison between Example 1 and Example 6 that when the esterification reaction is carried out in the presence of water, it is more conducive to the improvement of the yield of astaxanthin monoester.

[0096] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principle and spirit 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, wherein the organic carrier is a triblock copolymer, 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; The invention relates to a step of modifying and cross-linking an immobilized biological enzyme to obtain an immobilized magnetic carbonized lipase. The modification and cross-linking process comprises: subjecting the immobilized biological enzyme to a modification reaction with a modifier, wherein 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; subjecting the obtained modified immobilized biological enzyme to a cross-linking reaction with a cross-linking agent; washing and drying the obtained cross-linking reaction product, and obtaining the immobilized magnetic carbonized lipase.

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 claim 1, characterized in that: The modification and cross-linking conditions include: 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.

7. An immobilized biological enzyme prepared by the method according to any one of claims 1 to 6.

8. 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 7.

9. The method for synthesizing astaxanthin monoester according to claim 8, 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.

10. The method for synthesizing astaxanthin monoester according to claim 8, 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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