Magnetic microspheres and method for immobilizing DL-pantoate resolving enzyme

Abstract

The present invention provides magnetic microspheres for immobilizing DL-pantoate cleavage enzyme. These microspheres comprise a core composed of magnetic Fe3O4 / polymethyl methacrylate composite microspheres. Polyvinyl alcohol is grafted onto the surface of the core, and a sulfhydryl-containing group is attached to the polyvinyl alcohol. The present invention also provides a method for immobilizing DL-pantoate cleavage enzyme using the magnetic microspheres. The resulting immobilized enzyme exhibits high stability, high activity, and improved substrate binding.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a magnetic microsphere and a method for immobilizing DL-pantoate resolving enzyme using the magnetic microsphere. Background Art

[0002] D-pantoic acid lactone is an important chiral intermediate in the synthesis of D-pantoic acid products. Traditionally, D-pantoic acid lactone synthesis involves enzymatic hydrolysis of D-pantoic acid lactone from DL-pantoic acid lactone to produce L-pantoic acid lactone and D-pantoic acid. After separation of the acid ester, D-pantoic acid is lactonized to D-pantoic acid lactone. L-pantoic acid lactone is then chemically racemized to DL-pantoic acid lactone, and the above steps are repeated.

[0003] The enzyme immobilization method and carrier have a significant impact on the stability and catalytic activity of the immobilized enzyme. The physicochemical microenvironment provided by the immobilized enzyme carrier is a key factor in ensuring the efficient and stable enzyme. As an immobilized carrier, it must first possess significant reactive functional groups that can exhibit high stability in harsh reaction environments; second, it must have minimal steric hindrance, achieved by interacting with nucleophilic groups on the enzyme through bifunctional reagents or multi-point covalent linkages; third, it must be inert, able to establish a strong connection with biomolecules and prevent them from participating in the reaction; and fourth, it must possess a certain degree of physical strength, high porosity, and a large specific surface area.

[0004] Fe₃O₄ is a commonly used magnetic nanocarrier with advantages such as good biocompatibility, large specific surface area, strong surface modifiability, easy enzyme binding, and rapid and efficient recovery. Despite the numerous advantages of Fe₃O₄ nanoparticles as immobilization carriers, they also have limitations, such as high surface energy, susceptibility to aggregation, high chemical activity, susceptibility to oxidation, and easily damaged magnetic and dispersible properties. Therefore, it is necessary to design suitable carriers to enhance enzyme stability, improve catalytic efficiency, and minimize product inhibition.

[0005] Fe3O4 nanoparticles must be functionalized with some biocompatible compounds to enhance enzyme activity, stability and reusability. Summary of the Invention

[0006] The present invention aims to provide a method for immobilizing DL-pantoate cleavage enzyme. The present invention functionalizes Fe3O4 magnetic nanoparticles to obtain a carrier for immobilizing DL-pantoate cleavage enzyme. The use of the carrier can greatly improve the immobilization efficiency and recovery rate of DL-pantoate cleavage enzyme. The obtained immobilized enzyme has high activity, high stability, is easier to bind to the substrate, and is suitable for industrial production.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a magnetic microsphere having a magnetic Fe3O4 / polymethyl methacrylate composite microsphere as a core, polyvinyl alcohol being grafted onto the surface of the core, and a thiol-containing group being connected to the polyvinyl alcohol.

[0009] According to some embodiments of the present invention, the diameter of the magnetic microspheres is 200-250 nm.

[0010] According to some embodiments of the present invention, the average molecular weight of the polyvinyl alcohol is 20,000 to 100,000, preferably 65,000 to 75,000.

[0011] According to some embodiments of the invention, the polyvinyl alcohol is polyvinyl alcohol-7200.

[0012] The Fe3O4 / polymethyl methacrylate composite microspheres of the present invention can be obtained by a method comprising the following steps:

[0013] After mixing divalent iron salt and trivalent iron salt, oleic acid and ammonia water are added, and the mixture is reacted at 60℃ to 90℃ to obtain Fe3O4 magnetic nanoparticles;

[0014] The Fe3O4 magnetic nanoparticles are mixed with methyl methacrylate and divinylbenzene, and azobisisobutyronitrile and polyvinyl alcohol aqueous solution are added for emulsification, and the mixture is stirred for reaction to obtain magnetic Fe3O4 / polymethyl methacrylate composite microspheres.

[0015] In some embodiments, the Fe provided by the ferrous salt 2+ Fe provided by ferric salts 3+ The molar ratio is 1:(1-2), preferably 1:(1.2-1.8), more preferably 1:(1.4-1.6).

[0016] In some embodiments, the ferrous salt is ferrous sulfate heptahydrate or ferrous chloride tetrahydrate.

[0017] In some embodiments, the ferric salt is ferric chloride hexahydrate or ferric sulfate nonahydrate.

[0018] In some embodiments, the concentration of the aqueous ammonia is 20-25 wt %.

[0019] In some embodiments, the mass concentration of the polyvinyl alcohol aqueous solution is 1.0-1.8%.

[0020] In some embodiments, the polyvinyl alcohol is PVA17-88.

[0021] In some embodiments, the stirring speed is 300-400 r / min.

[0022] In some embodiments, the reaction temperature is 45-65°C, preferably 50-60°C.

[0023] In some embodiments, the reaction time is 4-6 hours.

[0024] According to some embodiments of the present invention, the preparation of the Fe3O4 / polymethyl methacrylate composite microspheres comprises the following steps:

[0025] (1) dissolving ferric chloride hexahydrate and ferrous sulfate heptahydrate in deionized water, adding oleic acid and 25% ammonia water, stirring at 60-85° C., and then magnetically separating to obtain a precipitate;

[0026] (2) mixing the obtained precipitate with oleic acid, stirring at 70-90° C. and 150-250 r / min for reaction, and then magnetically separating to obtain Fe 3 O 4 magnetic nanoparticles;

[0027] (3) The Fe3O4 magnetic nanoparticles obtained in step (2) are mixed with methyl methacrylate and divinylbenzene, and then azobisisobutyronitrile is added to dissolve the mixture, and then a polyvinyl alcohol aqueous solution is added. The mixture is stirred at 45-65°C and 300-400 r / min for 4-6 hours.

[0028] Preferably, after magnetic separation, the precipitate obtained in step (1) and the Fe3O4 magnetic nanoparticles obtained in step (2) are washed with deionized water.

[0029] Preferably, in step (3), the reaction is stirred at 45-54° C. for 50-70 min and then the temperature is raised to 55-65° C. and the stirring reaction is continued for 3.5-4.5 h.

[0030] In a second aspect, the present invention provides a method for preparing the magnetic microspheres according to the first aspect, comprising the following steps:

[0031] (1) Fe3O4 / polymethyl methacrylate composite microspheres are hydrolyzed under alkaline conditions, subjected to chlorination treatment, and then mixed with a solution containing polyvinyl alcohol to obtain magnetic microspheres with polyvinyl alcohol grafted on the surface;

[0032] (2) The magnetic microspheres with polyvinyl alcohol attached to the surface obtained in step (1) are mixed with a thiol-containing reagent to react, thereby obtaining magnetic microspheres with thiol groups attached to the polyvinyl alcohol.

[0033] According to some embodiments of the present invention, in step (1), the hydrolysis temperature is 40-60°C, preferably 45-55°C.

[0034] According to some embodiments of the present invention, in step (1), the hydrolysis time is 12 to 24 hours, preferably 14 to 20 hours.

[0035] According to a preferred embodiment of the present invention, in step (1), after the hydrolysis is completed, dilute hydrochloric acid is used to adjust the pH to 1-2, and then chlorination treatment is performed.

[0036] According to a more preferred embodiment of the present invention, in step (1), after the hydrolysis is completed, dilute hydrochloric acid is used to adjust the pH to 1-2, and the reaction is carried out for 10-30 minutes before acyl chlorination treatment.

[0037] According to some embodiments of the present invention, in step (1), a reagent containing thionyl chloride is used for chlorination treatment.

[0038] According to some embodiments of the present invention, in step (1), the polyvinyl alcohol solution is a polyvinyl alcohol N,N-dimethylformamide solution.

[0039] According to some embodiments of the present invention, in step (1), the reaction temperature is 40-60°C.

[0040] According to some embodiments of the present invention, in step (1), the reaction time is 2 to 4 hours.

[0041] According to some embodiments of the present invention, in step (1), the mass ratio of the Fe3O4 / polymethyl methacrylate composite microspheres to polyvinyl alcohol is 1:(5-10), for example, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0042] According to some embodiments of the present invention, the specific operations of step (1) include:

[0043] A. Mix Fe3O4 / polymethyl methacrylate composite microspheres with sodium hydroxide solution, hydrolyze at 40-60°C for 12-24 hours, adjust the pH to 1-2 with dilute hydrochloric acid, react at room temperature for 10-30 minutes, and wash with deionized water to remove impurities to obtain hydrolyzed composite microspheres;

[0044] B. mixing the hydrolyzed composite microspheres obtained in step A with dichloromethane, pyridine, a mixture of N,N-dimethylformamide and dichloromethane, and thionyl chloride, and reacting at room temperature for 1 to 3 hours to obtain chlorinated magnetic microspheres;

[0045] C. Mixing the chlorinated magnetic microspheres with pyridine and polyvinyl alcohol in N,N-dimethylformamide, stirring and reacting at 40-60° C. for 2-4 hours to obtain magnetic microspheres with surface grafted polyvinyl alcohol.

[0046] Preferably, in step A, the concentration of the sodium hydroxide solution is 2-3 mol / L.

[0047] Preferably, in step A, the hydrolysis is carried out with stirring at a rotation speed of 150 to 200 r / min.

[0048] Preferably, in step A, the stirring speed of the room temperature reaction is 140-160 r / min.

[0049] Preferably, in step B, the stirring speed of the room temperature reaction is 150-170 r / min.

[0050] Preferably, in step C, the concentration of the polyvinyl alcohol-N,N-dimethylformamide solution is 6-8% (w / v).

[0051] According to some embodiments of the present invention, in step (2), the mercapto-containing reagent is a mercaptosilane reagent, preferably 3-mercaptopropyltriethoxysilane.

[0052] According to some embodiments of the present invention, in step (2), the amount of the thiol-containing reagent is 3-4 times the amount of the hydroxyl substance on the surface of the magnetic microspheres grafted with polyvinyl alcohol.

[0053] According to some embodiments of the present invention, in step (2), the reaction temperature is 60-80°C, preferably 65-75°C.

[0054] According to some embodiments of the present invention, in step (2), the reaction time is 11 to 12 hours.

[0055] According to some embodiments of the present invention, in step (2), the reaction is carried out in the presence of a catalyst and an organic solvent.

[0056] In some embodiments, the catalyst is a titanate-based catalyst.

[0057] In some preferred embodiments, the catalyst is a tetraalkyl titanate.

[0058] In some more preferred embodiments, the catalyst is tetrabutyl titanate.

[0059] In some embodiments, the organic solvent is an aromatic organic solvent.

[0060] In some preferred embodiments, the organic solvent is benzene, toluene, or xylene.

[0061] In some more preferred embodiments, the organic solvent is xylene.

[0062] According to some embodiments of the present invention, the specific operations of step (2) include:

[0063] The magnetic microspheres with surface grafted polyvinyl alcohol obtained in step (1) are mixed with xylene, 3-mercaptopropyltriethoxysilane and tetrabutyl titanate, and reacted at 65-75° C. for 11-12 hours to obtain magnetic microspheres with thiol groups modified on polyvinyl alcohol.

[0064] According to a preferred embodiment of the present invention, the method for preparing the magnetic microspheres comprises the following steps:

[0065] (1) dissolving ferric chloride hexahydrate and ferrous sulfate heptahydrate in deionized water, adding oleic acid and ammonia water, stirring at 60-85°C, and then magnetically separating to obtain a precipitate, adding oleic acid to the precipitate, stirring at 70-90°C, and then magnetically separating to obtain Fe3O4 magnetic nanoparticles;

[0066] (2) mixing the Fe3O4 magnetic nanoparticles obtained in step (1) with methyl methacrylate and divinylbenzene, adding azobisisobutyronitrile to dissolve the mixture, adding a polyvinyl alcohol aqueous solution, reacting at 45-54°C for 50-70 minutes, then heating to 55-65°C and continuing the reaction for 3.5-4.5 hours to obtain Fe3O4 / polymethyl methacrylate composite microspheres;

[0067] (3) mixing the Fe3O4 / polymethyl methacrylate composite microspheres obtained in step (2) with a sodium hydroxide solution, hydrolyzing at 40-60°C for 12-24 hours, adjusting the pH to 1-2 with dilute hydrochloric acid, reacting at room temperature for 10-30 minutes, and washing with deionized water to remove impurities to obtain hydrolyzed composite microspheres;

[0068] (4) mixing the hydrolyzed composite microspheres obtained in step (3) with dichloromethane, pyridine, a mixture of N,N-dimethylformamide and dichloromethane, and thionyl chloride, and reacting at room temperature for 1 to 3 hours to obtain chlorinated magnetic microspheres;

[0069] (5) mixing the chlorinated magnetic microspheres obtained in step (4) with a solution of pyridine and polyvinyl alcohol in N,N-dimethylformamide, and reacting at 40-60° C. for 2-4 hours to obtain magnetic microspheres with surface grafted polyvinyl alcohol;

[0070] (6) The magnetic microspheres with surface grafted polyvinyl alcohol obtained in step (5) are mixed with xylene, 3-mercaptopropyltriethoxysilane, and tetrabutyl titanate, and reacted at 65-75° C. for 11-12 hours to obtain magnetic microspheres with thiol groups modified on the polyvinyl alcohol.

[0071] Preferably, the concentration of the ammonia water in step (1) is 20-25 wt%.

[0072] Preferably, after adding oleic acid in step (1), stirring at 70-90° C. and 150-250 r / min is performed, followed by magnetic separation.

[0073] Preferably, the reaction in step (2) is carried out by stirring at a rotation speed of 300-400 r / min.

[0074] Preferably, the concentration of the sodium hydroxide solution in step (3) is 2 to 3 mol / L.

[0075] Preferably, in step (3), the hydrolysis is carried out by stirring at a rotation speed of 150 to 200 r / min.

[0076] Preferably, the stirring speed of the room temperature reaction in step (3) is 140 to 160 r / min.

[0077] Preferably, the stirring speed of the room temperature reaction in step (4) is 150-170 r / min.

[0078] Preferably, the concentration of the polyvinyl alcohol-N,N-dimethylformamide solution in step (5) is 6-8% (w / v).

[0079] In a third aspect, the present invention provides an immobilized enzyme comprising DL-pantoate cleavage enzyme and the magnetic microspheres described in the first aspect of the present invention, or comprising DL-pantoate cleavage enzyme and the magnetic microspheres obtained by the preparation method described in the second aspect of the present invention.

[0080] Preferably, the mass ratio of the DL-pantoate resolving enzyme to the magnetic microspheres is (0.1-0.2):1, such as 0.12:1, 0.15:1, 0.16:1 or 0.18:1.

[0081] In a fourth aspect, the present invention provides a method for immobilizing DL-pantoate cleavage enzyme, wherein the DL-pantoate cleavage enzyme is immobilized using the magnetic microspheres described in the first aspect of the present invention or the magnetic microspheres prepared by the preparation method described in the second aspect as carriers.

[0082] According to some embodiments of the present invention, a cross-linking agent is further used in the immobilization method.

[0083] In some embodiments, the cross-linking agent contains a diepoxy group. Preferably, the cross-linking agent is polyethylene glycol diglycidyl ether.

[0084] The present invention uses Fe3O4 / polymethyl methacrylate composite microspheres grafted with polyvinyl alcohol and modified with thiol groups as immobilized enzyme carriers. The enzyme is adsorbed between polyvinyl alcohol chains through the interaction between the carrier and the DL-pantoate cleavage enzyme. The diepoxy groups of the cross-linking agent react with the thiol groups modified on the polyvinyl alcohol chains to encapsulate the DL-pantoate cleavage enzyme on the surface of the magnetic microspheres.

[0085] According to some embodiments of the present invention, the method comprises the steps of:

[0086] S1. The magnetic microspheres according to the first aspect of the present invention or the magnetic microspheres prepared by the preparation method according to the second aspect of the present invention are mixed with DL-pantoate resolving enzyme, sealed, and incubated for 2 to 4 hours to obtain a loaded enzyme solution;

[0087] S2. The enzyme solution obtained in step S1 is mixed with a cross-linking agent, sealed, and incubated for 0.5 to 1 hour to obtain immobilized DL-pantoate resolving enzyme.

[0088] Preferably, in step S1, the mixture is incubated with shaking at a rotation speed of 180 to 200 r / min for 2 to 4 hours.

[0089] Preferably, in step S1, the mass ratio of the DL-pantoate resolving enzyme to the magnetic microspheres is (0.1-0.2):1, such as 0.12:1, 0.15:1, 0.16:1 or 0.18:1.

[0090] Preferably, in step S2, the mixture is incubated with shaking at a rotation speed of 150 to 200 r / min for 0.5 to 1 h.

[0091] Specifically, the method comprises the following steps:

[0092] A. mixing the magnetic microspheres described in the first aspect of the present invention or the magnetic microspheres prepared by the preparation method described in the second aspect with PBS buffer to obtain swollen magnetic microspheres;

[0093] B. Add DL-pantoate resolving enzyme to the swollen magnetic microspheres, shake at 180-200 rpm at room temperature, incubate for 2-4 hours, and then magnetically separate to obtain the loaded enzyme solution;

[0094] C. The loaded enzyme solution was thoroughly mixed with polyethylene glycol diglycidyl ether, then sealed with nitrogen and incubated at room temperature with shaking at 150-200 rpm for 0.4-1 h to obtain immobilized DL-pantoate resolving enzyme loaded on magnetic microspheres.

[0095] In some embodiments, the molecular weight of the polyethylene glycol diglycidyl ether is 150-600, preferably 350-400.

[0096] In some embodiments, the polyethylene glycol diglycidyl ether is diluted using PBS buffer.

[0097] In some embodiments, the pH of the PBS buffer is 6.8-7.2.

[0098] In some embodiments, the concentration of the PBS buffer is 20-30 mM.

[0099] In a fifth aspect, the present invention provides use of the magnetic microspheres described in the first aspect or the magnetic microspheres obtained by the preparation method described in the second aspect in immobilizing enzymes.

[0100] According to some embodiments of the invention, the enzyme is a DL-pantoate resolvase.

[0101] According to some embodiments of the present invention, the magnetic microspheres serve as immobilized enzyme carriers.

[0102] Compared with the prior art, the present invention has the following beneficial effects: the present invention uses Fe3O4 / polymethyl methacrylate composite microspheres grafted with polyvinyl alcohol and modified with thiol groups as immobilized enzyme carriers, adsorbs DL-pantoate cleavage enzyme between polyvinyl alcohol chains, and encapsulates DL-pantoate cleavage enzyme on the surface of magnetic microspheres by reacting the diepoxy groups of the cross-linking agent with the thiol groups modified on the polyvinyl alcohol chains. The active conformation of the enzyme is not destroyed, the enzyme is firmly immobilized, and the stability and activity of the enzyme can be maintained for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Figure 1 The desorption results of the immobilized enzyme after loading and encapsulation in Examples 1-3 and Comparative Examples 1-2 are shown.

[0104] Figure 2 The stability test results of the immobilized enzymes in Examples 1-3 and Comparative Examples 1-2 are shown. DETAILED DESCRIPTION

[0105] The following specific embodiments illustrate the implementation of the present invention. People familiar with this technology can easily understand other advantages and functions of the present invention from the content disclosed in this specification. The following embodiments are provided for a better understanding of the present invention, but are not intended to limit the present invention.

[0106] Unless otherwise specified, the experimental methods in the following examples are all conventional methods. Unless otherwise specified, the experimental materials used in the following examples are all purchased from conventional biochemical reagent stores.

[0107] Example 1:

[0108] This embodiment provides a method for immobilizing DL-pantoate cleavage enzyme, which specifically includes the following steps:

[0109] 1. Preparation of magnetic microspheres

[0110] (1) 5.406g of ferric chloride hexahydrate and 10.008g of ferrous sulfate heptahydrate (Fe 3+ :Fe 2+ =1:1.8) were added to 100 mL of deionized water and ultrasonically stirred until completely dissolved. 2 mL of oleic acid was added, followed by the dropwise addition of 20 mL of 25% ammonia. The mixture was mechanically stirred at 300 rpm in a 60°C water bath for 40 min, followed by magnetic separation and rinsing with deionized water. An appropriate amount of oleic acid was added to the precipitate, which was mechanically stirred at 200 rpm in an 80°C water bath for 40 min, followed by magnetic separation, rinsing with deionized water, and drying at 50°C to obtain Fe3O4 magnetic nanoparticles.

[0111] (2) Weigh 100 mg of Fe3O4 magnetic nanoparticles, add 3.8 mL of methyl methacrylate and 280 μL of divinylbenzene, and disperse evenly until fully swollen. Add 140 mg of azobisisobutyronitrile, dissolve it by ultrasonication, and then add it to 17 mL of a 1.3% aqueous solution of polyvinyl alcohol-1788. After ultrasonic emulsification, stir mechanically at 50°C and 400 rpm for 2 h, then heat to 60°C and react for 3 h. Magnetic separation precipitates are performed, washed with deionized water, and dried at 50°C to obtain Fe3O4 / polymethyl methacrylate composite microspheres.

[0112] (3) Weigh 500 mg of the magnetic microspheres obtained in step (2) and add 60 mL of 2 M sodium hydroxide solution. Esterify at 40°C, 150 rpm for 12 h. Adjust the pH to 1.0 with 1 M dilute hydrochloric acid and shake at 150 rpm for 20 min at room temperature. Magnetic separation precipitates are performed, washed with deionized water, and dried at 50°C for later use.

[0113] (4) Weigh 1 g of the magnetic microspheres obtained in step (3), add 50 mL of dichloromethane, 122 μL of pyridine, 200 μL of a mixture of N,N-dimethylformamide and dichloromethane (1:99, v / v), and 75 μL of thionyl chloride in sequence, and shake at 40°C, 160 rpm for 2 h. Continue adding 122 μL of pyridine, and then add 64 mL of a solution of polyvinyl alcohol-7200 in N,N-dimethylformamide (6%, w / v), and stir magnetically at 50°C for 2 h. Separate the precipitate by magnetic separation, wash with deionized water, and dry at 50°C to obtain magnetic microspheres with surface grafted polyvinyl alcohol.

[0114] (5) Weigh 300 mg of the magnetic microspheres obtained in step (4), add 20 mL of xylene and 2.25 μL of tetrabutyl titanate, and then add 800 μL of 3-mercaptopropyltriethoxysilane. Reflux the mixture in a nitrogen-protected water bath at 70°C for 11 h. Magnetic separation and precipitation are performed, the mixture is washed with deionized water, and dried at 50°C to obtain magnetic microspheres with surface modified thiol groups.

[0115] 2. Immobilization of DL-pantoate cleavage enzyme using magnetic microspheres

[0116] Weigh 100 mg of the magnetic microspheres obtained in step (5) above, add 10 mL of PBS buffer (pH 7.0, 20 mM) to fully swell, and then magnetically separate. Add 8 mL of 2 mg / mL DL-pantoate splitting enzyme solution, protect with nitrogen, incubate in a shaker at room temperature at 180 r / min for 2 h, and then magnetically separate. Add 10 mL of PBS buffer (pH 7.0, 20 mM), and then add 50 μL of polyethylene glycol diglycidyl ether diluted 400 times with pH 7.0, 20 mM PBS buffer, protect with nitrogen, incubate in a shaker at room temperature at 180 r / min for 0.5 h, and then magnetically separate. The enzyme loading capacity was determined to be 40 mg / g ( Figure 1 a), the resolution rate was 11.1%, and the catalytic relative enzyme activity was maintained at 97% of the initial enzyme activity for 10 consecutive batches ( Figure 2 a).

[0117] Example 2:

[0118] This embodiment provides a method for immobilizing DL-pantoate cleavage enzyme, which specifically includes the following steps:

[0119] 1. Preparation of magnetic microspheres

[0120] (1) 5.406g of ferric chloride hexahydrate and 6.672g of ferrous sulfate heptahydrate (Fe 3+ :Fe 2+ =1:1.2) were added to 100 mL of deionized water and ultrasonically stirred until completely dissolved. 1.5 mL of oleic acid was added, followed by 20 mL of 20% ammonia solution. The mixture was mechanically stirred at 300 rpm in a 60°C water bath for 40 min, followed by magnetic separation and rinsing with deionized water. An appropriate amount of oleic acid was added to the precipitate, which was mechanically stirred at 200 rpm in an 80°C water bath for 40 min, followed by magnetic separation, rinsing with deionized water, and drying at 50°C to obtain Fe3O4 magnetic nanoparticles.

[0121] (2) Weigh 100 mg of Fe3O4 magnetic nanoparticles, add 3.6 mL of methyl methacrylate and 270 μL of divinylbenzene, and disperse evenly until fully swollen. Add 136 mg of azobisisobutyronitrile, dissolve it by ultrasonication, and then add it to 17 mL of a 1.5% aqueous solution of polyvinyl alcohol-1788. After ultrasonic emulsification, stir mechanically at 50°C and 400 rpm for 2 h, then heat to 65°C and react for 3 h. Separate the precipitate by magnetic separation, wash with deionized water, and dry at 50°C to obtain Fe3O4 / polymethyl methacrylate composite microspheres.

[0122] (3) Weigh 500 mg of the magnetic microspheres obtained in step (2) and add 40 mL of 3 M sodium hydroxide solution. Esterify at 45°C, 150 rpm for 15 h. Adjust the pH to 1.5 with 1 M dilute hydrochloric acid and shake at 150 rpm for 20 min at room temperature. Magnetic separation precipitates are performed, washed with deionized water, and dried at 50°C for later use.

[0123] (4) Weigh 1 g of the magnetic microspheres obtained in step (3), add 50 mL of dichloromethane, 122 μL of pyridine, 200 μL of a mixture of N,N-dimethylformamide and dichloromethane (1:99, v / v), and 75 μL of thionyl chloride in sequence, and shake at 40°C, 160 rpm for 2 h. Continue adding 122 μL of pyridine, and then add 64 mL of a solution of polyvinyl alcohol-7200 in N,N-dimethylformamide (6%, w / v), and stir magnetically at 50°C for 2 h. Separate the precipitate by magnetic separation, wash with deionized water, and dry at 50°C to obtain magnetic microspheres with surface grafted polyvinyl alcohol.

[0124] (5) Weigh 300 mg of the magnetic microspheres obtained in step (4), add 20 mL of xylene and 2.25 μL of tetrabutyl titanate, and then add 800 μL of 3-mercaptopropyltriethoxysilane. Reflux the mixture in a nitrogen-protected water bath at 65°C for 12 h. Magnetic separation and precipitation are performed, the mixture is washed with deionized water, and dried at 50°C to obtain magnetic microspheres with surface modified thiol groups.

[0125] 2. Immobilization of DL-pantoate cleavage enzyme using magnetic microspheres

[0126] Weigh 100 mg of the magnetic microspheres obtained in step (5) above, add 10 mL of PBS buffer (pH 7.0, 20 mM) to fully swell, and then magnetically separate. Add 8 mL of 2 mg / mL DL-pantoate splitting enzyme solution, protect with nitrogen, incubate in a shaker at 180 r / min at room temperature for 3 h, and then magnetically separate. Add 10 mL of PBS buffer (pH 7.0, 20 mM), and then add 50 μL of polyethylene glycol diglycidyl ether diluted 400 times with pH 7.0, 20 mM PBS buffer, protect with nitrogen, incubate in a shaker at 160 r / min at room temperature for 1 h, and then magnetically separate. The enzyme immobilization capacity was determined to be 44 mg / g ( Figure 1 b), the resolution rate was 13.7%, and the catalytic relative enzyme activity was maintained at 95% of the initial enzyme activity for 10 consecutive batches ( Figure 2 b).

[0127] Example 3:

[0128] This embodiment provides a method for immobilizing DL-pantoate cleavage enzyme, which specifically includes the following steps:

[0129] 1. Preparation of magnetic microspheres

[0130] (1) 5.406g of ferric chloride hexahydrate and 8.340g of ferrous sulfate heptahydrate (Fe 3+ :Fe 2+=1:1.5) were added to 100 mL of deionized water and ultrasonically stirred until completely dissolved. 2.5 mL of oleic acid was then added dropwise, followed by 20 mL of 25% ammonia water. The mixture was mechanically stirred at 300 rpm in a 60°C water bath for 40 min, followed by magnetic separation and rinsing with deionized water. An appropriate amount of oleic acid was added to the precipitate, which was mechanically stirred at 200 rpm in an 80°C water bath for 40 min, followed by magnetic separation, rinsing with deionized water, and drying at 50°C to obtain Fe3O4 magnetic nanoparticles.

[0131] (2) Weigh 100 mg of Fe3O4 magnetic nanoparticles, add 3.8 mL of methyl methacrylate and 280 μL of divinylbenzene, and disperse evenly until fully swollen. Add 140 mg of azobisisobutyronitrile, dissolve it by ultrasonication, and then add it to 17 mL of a 1.8% aqueous solution of polyvinyl alcohol-1788. After ultrasonic emulsification, stir mechanically at 50°C and 400 rpm for 1 hour, then heat to 60°C and react for 4 hours. Separate the precipitate by magnetic separation, wash with deionized water, and dry at 50°C to obtain Fe3O4 / polymethyl methacrylate composite microspheres.

[0132] (3) Weigh 500 mg of the magnetic microspheres obtained in step (2) and add 60 mL of 2 M sodium hydroxide solution. Esterify at 50°C, 200 rpm for 16 h. Adjust the pH to 1.0 with 1 M dilute hydrochloric acid and shake at 200 rpm for 20 min at room temperature. Magnetic separation precipitates are performed, washed with deionized water, and dried at 50°C for later use.

[0133] (4) Weigh 1 g of the magnetic microspheres obtained in step (3) and add 50 mL of dichloromethane, 122 μL of pyridine, 200 μL of a mixture of N,N-dimethylformamide and dichloromethane (1:99, v / v), and 75 μL of thionyl chloride in sequence. Shake at 40°C and 160 rpm for 2 h. Add 122 μL of pyridine and then 64 mL of a solution of polyvinyl alcohol-7200 in N,N-dimethylformamide (6%, w / v). Stir magnetically at 50°C for 2 h. Separate the precipitate by magnetic separation, wash with deionized water, and dry at 50°C for later use.

[0134] (5) Weigh 300 mg of the magnetic microspheres obtained in step (4), add 20 mL of xylene and 2.25 μL of tetrabutyl titanate, and then add 800 μL of 3-mercaptopropyltriethoxysilane. Reflux the mixture in a nitrogen-protected water bath at 70°C for 11.5 h. Magnetic separation and precipitation are performed, the mixture is washed with deionized water, and dried at 50°C to obtain magnetic microspheres with surface modified thiol groups.

[0135] 2. Immobilization of DL-pantoate cleavage enzyme using magnetic microspheres

[0136] Weigh 100 mg of the magnetic microspheres prepared in step (5) above, add 10 mL of PBS buffer (pH 7.0, 20 mM) to fully swell, and then magnetically separate. Add 8 mL of 2 mg / mL DL-pantoate splitting enzyme solution, protect with nitrogen, incubate in a shaker at room temperature at 190 r / min for 3 h, and then magnetically separate. Add 10 mL of PBS buffer (pH 7.0, 20 mM), and then add 50 μL of polyethylene glycol diglycidyl ether diluted 400 times with pH 7.0, 20 mM PBS buffer, protect with nitrogen, incubate in a shaker at room temperature at 180 r / min for 0.5 h, and then magnetically separate. The enzyme immobilization capacity was determined to be 65 mg / g ( Figure 1 c), the resolution rate was 7.1%, and the catalytic relative enzyme activity was maintained at 98% of the initial enzyme activity for 10 consecutive batches ( Figure 2 c).

[0137] Comparative Example 1

[0138] This comparative example provides a method for immobilizing DL-pantoate cleavage enzyme, which differs from Example 1 only in that the prepared magnetic microspheres are not modified with polyvinyl alcohol and thiol groups. The method specifically comprises the following steps:

[0139] 1. Preparation of magnetic microspheres

[0140] (1) 5.406g of ferric chloride hexahydrate and 10.008g of ferrous sulfate heptahydrate (Fe 3+ :Fe 2+ =1:1.8) were added to 100 mL of deionized water and ultrasonically stirred until completely dissolved. 2 mL of oleic acid was added, followed by the dropwise addition of 20 mL of 25% ammonia. The mixture was mechanically stirred at 300 rpm in a 60°C water bath for 40 min, followed by magnetic separation and rinsing with deionized water. An appropriate amount of oleic acid was added to the precipitate, which was mechanically stirred at 200 rpm in an 80°C water bath for 40 min, followed by magnetic separation, rinsing with deionized water, and drying at 50°C to obtain Fe3O4 magnetic nanoparticles.

[0141] (2) Weigh 100 mg of Fe3O4 magnetic nanoparticles, add 3.8 mL of methyl methacrylate and 280 μL of divinylbenzene, and disperse evenly until fully swollen. Add 140 mg of azobisisobutyronitrile, dissolve it by ultrasonication, and then add it to 17 mL of a 1.3% aqueous solution of polyvinyl alcohol-1788. After ultrasonic emulsification, stir mechanically at 50°C and 400 rpm for 2 h, then heat to 60°C and react for 3 h. Magnetic separation precipitates are performed, washed with deionized water, and dried at 50°C to obtain Fe3O4 / polymethyl methacrylate composite microspheres.

[0142] 2. Immobilization of DL-pantoate cleavage enzyme using magnetic microspheres

[0143] Weigh 100 mg of the Fe3O4 / polymethyl methacrylate composite microspheres obtained in step (2) above, add 10 mL of PBS buffer (pH 7.0, 20 mM) to fully swell, and then magnetically separate. Add 8 mL of 2 mg / mL DL-pantoate splitting enzyme solution, protect with nitrogen, incubate in a shaker at room temperature at 180 r / min for 2 h, and then magnetically separate. Add 10 mL of PBS buffer (pH 7.0, 20 mM), and then add 50 μL of polyethylene glycol diglycidyl ether diluted 400 times with pH 7.0, 20 mM PBS buffer, protect with nitrogen, incubate in a shaker at room temperature at 180 r / min for 0.5 h, and then magnetically separate. The enzyme loading capacity was determined to be 35 mg / g ( Figure 1 d), the resolution rate was 50%, and the catalytic relative enzyme activity was maintained at 41% of the initial enzyme activity for 10 consecutive batches ( Figure 2 d).

[0144] Comparative Example 2

[0145] This comparative example provides a method for immobilizing DL-pantoate cleavage enzyme, which differs from Example 1 only in that the prepared magnetic microspheres are not modified with thiol groups. The method specifically comprises the following steps:

[0146] 1. Preparation of magnetic microspheres

[0147] (1) 5.406g of ferric chloride hexahydrate and 10.008g of ferrous sulfate heptahydrate (Fe 3+ :Fe 2+ =1:1.8) were added to 100 mL of deionized water and ultrasonically stirred until completely dissolved. 2 mL of oleic acid was added, followed by the dropwise addition of 20 mL of 25% ammonia. The mixture was mechanically stirred at 300 rpm in a 60°C water bath for 40 min, followed by magnetic separation and rinsing with deionized water. An appropriate amount of oleic acid was added to the precipitate, which was mechanically stirred at 200 rpm in an 80°C water bath for 40 min, followed by magnetic separation, rinsing with deionized water, and drying at 50°C to obtain Fe3O4 magnetic nanoparticles.

[0148] (2) Weigh 100 mg of Fe3O4 magnetic nanoparticles, add 3.8 mL of methyl methacrylate and 280 μL of divinylbenzene, and disperse evenly until fully swollen. Add 140 mg of azobisisobutyronitrile, dissolve it by ultrasonication, and then add it to 17 mL of a 1.3% aqueous solution of polyvinyl alcohol-1788. After ultrasonic emulsification, stir mechanically at 50°C and 400 rpm for 2 h, then heat to 60°C and react for 3 h. Magnetic separation precipitates are performed, washed with deionized water, and dried at 50°C to obtain Fe3O4 / polymethyl methacrylate composite microspheres.

[0149] (3) Weigh 500 mg of the magnetic microspheres obtained in step (2) and add 60 mL of 2 M sodium hydroxide solution. Esterify at 40°C, 150 rpm for 12 h. Adjust the pH to 1.0 with 1 M dilute hydrochloric acid and shake at 150 rpm for 20 min at room temperature. Magnetic separation precipitates are performed, washed with deionized water, and dried at 50°C for later use.

[0150] (4) Weigh 1 g of the magnetic microspheres obtained in step (3), add 50 mL of dichloromethane, 122 μL of pyridine, 200 μL of a mixture of N,N-dimethylformamide and dichloromethane (1:99, v / v), and 75 μL of thionyl chloride in sequence, and shake at 40°C, 160 rpm for 2 h. Continue adding 122 μL of pyridine, and then add 64 mL of a solution of polyvinyl alcohol-7200 in N,N-dimethylformamide (6%, w / v), and stir magnetically at 50°C for 2 h. Separate the precipitate by magnetic separation, wash with deionized water, and dry at 50°C to obtain magnetic microspheres with surface grafted polyvinyl alcohol.

[0151] 2. Immobilization of DL-pantoate cleavage enzyme using magnetic microspheres

[0152] Weigh 100 mg of the magnetic microspheres obtained in step (4) above, add 10 mL of PBS buffer (pH 7.0, 20 mM) to fully swell, and then magnetically separate. Add 8 mL of 2 mg / mL DL-pantoate splitting enzyme solution, protect with nitrogen, incubate in a shaker at 180 r / min at room temperature for 2 h, and then magnetically separate. Add 10 mL of PBS buffer (pH 7.0, 20 mM), and then add 50 μL of polyethylene glycol diglycidyl ether diluted 400 times with pH 7.0, 20 mM PBS buffer, protect with nitrogen, incubate in a shaker at 180 r / min at room temperature for 0.5 h, and then magnetically separate. The enzyme immobilization capacity was determined to be 38 mg / g ( Figure 1 e), the resolution rate was 38.8%, and the catalytic relative enzyme activity was maintained at 52% of the initial enzyme activity for 10 consecutive batches ( Figure 2 e).

[0153] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A method for immobilizing DL-pantoate cleavage enzyme, comprising immobilizing the DL-pantoate cleavage enzyme using magnetic microspheres as carriers, wherein the magnetic microspheres have a core composed of magnetic Fe3O4 / polymethyl methacrylate composite microspheres, the surface of the core being grafted with polyvinyl alcohol, and the polyvinyl alcohol being connected to a thiol-containing group; The method comprises the following steps: S1. Mix the magnetic microspheres with DL-pantoate resolvase, seal the solution, and incubate for 2–4 h to obtain the loaded enzyme solution. S2. The enzyme solution obtained in step S1 was mixed with a cross-linking agent and sealed, and incubated for 0.5 to 1 h to obtain immobilized DL-pantoate resolving enzyme; in, The cross-linking agent is polyethylene glycol diglycidyl ether; The mass ratio of the DL-pantoate splitting enzyme to the magnetic microspheres is (0.1-0.2):

1.

2. The immobilization method according to claim 1, characterized in that The diameter of the magnetic microspheres is 200-250 nm; and / or the average molecular weight of the polyvinyl alcohol is 20,000-100,000.

3. The immobilization method according to claim 2, characterized in that The average molecular weight of the polyvinyl alcohol is 65,000-75,000.

4. The immobilization method according to any one of claims 1 to 3, characterized in that The preparation method of magnetic microspheres comprises the following steps: (1) Fe3O4 / polymethyl methacrylate composite microspheres are hydrolyzed under alkaline conditions, subjected to chlorination treatment, and then mixed with a solution containing polyvinyl alcohol to obtain magnetic microspheres with surface grafted polyvinyl alcohol; (2) The magnetic microspheres with surface grafted polyvinyl alcohol obtained in step (1) are mixed and reacted with a thiol-containing reagent to obtain magnetic microspheres with thiol groups attached to polyvinyl alcohol.

5. The immobilization method according to claim 4, characterized in that In step (1), after the hydrolysis is completed, dilute hydrochloric acid is used to adjust the pH to 1-2, and then chlorination treatment is performed.

6. The immobilization method according to claim 4, wherein In step (1), the hydrolysis temperature is 40-60°C and the hydrolysis time is 12-24 h; and / or in step (1), using a reagent containing thionyl chloride to carry out chlorination treatment; And / or in step (1), the reaction temperature is 40-60°C, and the reaction time is 2-4 h; And / or in step (1), the mass ratio of the Fe3O4 / polymethyl methacrylate composite microspheres to polyvinyl alcohol is 1:(5-10).

7. The immobilization method according to claim 6, characterized in that In step (1), the hydrolysis temperature is 45-55°C, and the hydrolysis time is 14-20 h.

8. The immobilization method according to claim 4, characterized in that In step (2), the thiol-containing reagent is a mercaptosilane reagent; And / or in step (2), the reaction temperature is 65-75° C., and the reaction time is 11-12 h.

9. The immobilization method according to claim 8, characterized in that In step (2), the mercapto-containing reagent is 3-mercaptopropyltriethoxysilane.

10. The immobilization method according to claim 4, characterized in that: In step (2), the reaction is carried out in the presence of a catalyst and an organic solvent.

11. The immobilization method according to claim 10, characterized in that: The catalyst is a titanate catalyst.

12. The immobilization method according to claim 10, characterized in that The catalyst is a tetraalkyl titanate.

13. The immobilization method according to claim 10, characterized in that The catalyst is tetrabutyl titanate.

14. The immobilization method according to claim 10, characterized in that The organic solvent is an aromatic organic solvent.

15. The immobilization method according to claim 10, characterized in that The organic solvent is benzene, toluene or xylene.

16. The immobilization method according to any one of claims 1 to 4, characterized in that: The preparation of the Fe3O4 / polymethyl methacrylate composite microspheres comprises the following steps: After mixing divalent iron salt and trivalent iron salt, oleic acid and ammonia water were added and reacted at 60℃~85℃ to obtain Fe3O4 magnetic nanoparticles; The Fe3O4 magnetic nanoparticles are mixed with methyl methacrylate and divinylbenzene, and azobisisobutyronitrile and polyvinyl alcohol aqueous solution are added for emulsification, and magnetic Fe3O4 / polymethyl methacrylate composite microspheres are obtained after reaction.

17. The immobilization method according to claim 16, characterized in that: The Fe provided by the divalent iron salt 2+ Fe provided by ferric salts 3+ The molar ratio is 1: (1~2); the mass concentration of the polyvinyl alcohol aqueous solution is 1.0~1.8%.

18. The immobilization method according to claim 16, characterized in that: The Fe provided by the divalent iron salt 2+ Fe provided by ferric salts 3+ The molar ratio is 1:(1.2~1.8).

19. The immobilization method according to claim 16, wherein: The reaction temperature is 45-65° C., and the reaction time is 4-6 h.

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