Chitosan microspheres as well as preparation and application thereof

Abstract

The invention belongs to the field of nano materials, and particularly relates to a preparation method of chitosan microspheres, which comprises the following steps: obtaining a water phase W1 dissolved with chitosan, and then adding an organic phase O1 containing a first emulsifier and a pore-foaming agent into the water phase W1 to form an O1 / W1 type emulsion; the pore-foaming agent is a hydrophobic component; (2) preparing an organic phase O2 in which a hydrophobic solvent and a second emulsifier are dissolved, and adding the O1 / W1 type emulsion obtained in the step (1) into the organic phase O2 to form an O1 / W1 / O2 type emulsion; carrying out pre-reaction on the O1 / W1 / O2 type emulsion and unit aldehyde, and then mixing with a cross-linking agent for cross-linking to obtain cross-linked chitosan; the preparation method comprises the following steps: carrying out acid treatment on cross-linked chitosan in advance, then carrying out alkali treatment, and activating the cross-linked chitosan and a dialdehyde component to prepare the chitosan microspheres. The invention also comprises the chitosan microspheres prepared by the preparation method and application of the chitosan microspheres in active ingredient loading. The material provided by the invention has excellent active component loading capacity, loading activity and stability.

Description

Technical Field

[0001] The invention belongs to the field of natural polymers, and in particular relates to the field of preparation of chitosan microspheres. Background Art

[0002] Chitosan, also known as glucosamine, is a linear polysaccharide composed of amino-D-glucose and N-acetyl-D-glucosamine linked by β-(1,4) glycosidic bonds. After dissolving, it can be made into different materials, such as sponges, gels, scaffolds and nanofibers. At the same time, chitosan has the advantages of good mechanical strength, antibacterial properties, protein affinity, biodegradability, etc. It is an ideal material that can be used in medicine, food, agriculture, textile industry and water treatment. In addition, chitosan molecules contain rich functional groups, such as amino and hydroxyl groups, and are easy to undergo chemical reactions, such as Schiff base, carboxymethylation, acylation, quaternization, esterification and other reactions.

[0003] In the biopharmaceutical industry, chitosan is mostly used to prepare microspheres or microcapsules, mainly because chitosan has antibacterial, adhesive and affinity characteristics. Microcapsules are mainly used for drug embedding and sustained release to prevent drugs from being destroyed by the digestive system and to prolong the retention time of drugs in the body. Microsphere carriers can be used for metal ion adsorption, protein adsorption and cell culture.

[0004] At present, chitosan microspheres are often prepared by emulsification crosslinking and ion precipitation. For example, Chinese patent document No. CN104324700A discloses a method for preparing a high-amino porous chitosan resin: (1) dissolving chitosan in a 2% by mass acetic acid solution to prepare a chitosan solution, and stirring at 50°C until it is completely dissolved; (2) adding liquid paraffin and a porogen by a reverse suspension method, and stirring sufficiently to make the reaction system into an emulsion; (3) adding formaldehyde solution to the mixed system and pre-crosslinking at 60°C for 40 to 60 minutes; (4) adjusting the pH of the solution to 10 to 13 with sodium hydroxide, adding epichlorohydrin solution, and reacting at 80°C for 3 hours; (5) filtering, washing with water, extracting, and then washing with acetone and ethanol; (6) then placing in a 0.5 mol / L hydrochloric acid solution, stirring and filtering, and finally soaking in a 1.0 mol / L sodium hydroxide solution, washing with water until neutral, to obtain a high-amino porous chitosan resin. The beneficial effects are good adsorption effect and reusability.

[0005] For another example, a Chinese patent document with publication number CN102652833A discloses a gastric targeted drug carrier and a preparation method thereof. The specific preparation method disclosed is: 1) Preparation of a drug-containing emulsion: using an aqueous solution of chitosan, chitosan quaternary ammonium salt and sodium glycerol phosphate as the aqueous phase W, and preparing a drug-containing emulsion with a drug and an oil phase containing an emulsifier; 2) heating the drug-containing emulsion in step 1) and stirring it to cross-link the chitosan quaternary ammonium salt and sodium glycerol phosphate contained therein to obtain a gel microsphere suspension, heating it and adding a cross-linking agent to cause a second cross-linking to solidify the gel microspheres.

[0006] In summary, although the prior art discloses some contents of emulsion cross-linked chitosan materials, the immobilization stability of cross-linked chitosan prepared by the prior method in enzyme immobilization and the activity of immobilized enzymes still have room for improvement. Summary of the invention

[0007] In view of the problems existing in the prior art, the first object of the present invention is to provide a method for preparing chitosan microspheres, aiming to prepare chitosan microspheres with excellent enzyme immobilization capacity, immobilization activity and stability.

[0008] The second purpose of the present invention is to provide chitosan microspheres prepared by the preparation method and use thereof as carriers for loading active ingredients.

[0009] The third object of the present invention is to provide an immobilized enzyme (cured enzyme) immobilized by the chitosan microspheres and its preparation method.

[0010] A method for preparing chitosan microspheres, comprising the steps of:

[0011] Step (1): Emulsion preparation

[0012] Obtaining an aqueous phase W1 in which chitosan is dissolved, and then adding an organic phase O1 containing a first emulsifier and a porogen to the aqueous phase W1 to form an O1 / W1 type emulsion; the porogen is a hydrophobic component;

[0013] Obtaining an organic phase O2 in which a hydrophobic solvent and a second emulsifier are dissolved, and adding the O1 / W1 type emulsion of step (1) to the organic phase O2 to form an O1 / W1 / O2 type emulsion;

[0014] Step (2): Emulsion cross-linking

[0015] The O1 / W1 / O2 type emulsion and the unit aldehyde are pre-reacted, and then mixed with a cross-linking agent for cross-linking to obtain cross-linked chitosan;

[0016] Step (3): Modification

[0017] The cross-linked chitosan is treated with acid in advance, then treated with alkali, and then activated with dialdehyde components to obtain the chitosan microspheres.

[0018] The present invention innovatively prepares chitosan to form an O1 / W1 / O2 emulsion, then performs a cross-linking treatment, and further pre-treats the cross-linked chitosan with acid first and then alkali and activates it with dialdehyde, so that the surface structure and active sites of the material can be optimized, so that it can adapt to the requirements of enzyme immobilization, improve its enzyme immobilization rate, avoid the attenuation of the activity of the immobilized enzyme, and improve its enzyme activity cyclic stability and high temperature resistance.

[0019] The present invention shows that pre-forming chitosan into an O / W emulsion and further combining the porogen and subsequent processes can further achieve synergy and enhance the chitosan microspheres' enzyme immobilization capacity, immobilization stability and immobilized enzyme activity stability.

[0020] In the present invention, the aqueous phase W1 is an acid aqueous solution in which chitosan is dissolved; further, it can be an acetic acid aqueous solution in which chitosan is dissolved.

[0021] In the present invention, the concentration of chitosan in the aqueous phase W1 has no particular requirement, and can be, for example, 0.01 to 0.1 g / mL, and further can be 0.02 to 0.05 g / mL;

[0022] In the present invention, the first emulsifier may be a conventional dispersant for forming an emulsion in the industry, for example, may include at least one of tween 80 and tween 60;

[0023] In the present invention, the porogen includes at least one of hydrophobic hydrocarbons and esters, and further at least one of alkanes, cycloalkanes, olefins, aromatic hydrocarbons, and ethyl acetate with a carbon number of less than 10, and further can be at least one of cyclopentane, cyclohexane, cycloheptane, n-heptane, and n-hexane.

[0024] In the present invention, in the organic phase O1, the volume ratio of the first emulsifier to the porogen is 1:1-15; further, it can be 1:3-10;

[0025] In the present invention, the volume ratio of the aqueous phase W1 to the organic phase O1 is 100:15-25; further 100:19-22.

[0026] In the present invention, chitosan is formed into an O1 / W1 emulsion in advance and then into an O1 / W1 / O2 emulsion, which can be combined with subsequent processes to optimize the physicochemical structure of chitosan, further synergistically improve its adaptability to enzyme immobilization, and improve enzyme immobilization effect and enzyme activity.

[0027] In the present invention, the second emulsifier may be an emulsifier known in the industry that can form an emulsion, for example, at least one of span 80, span 60 and AEO-3;

[0028] In the present invention, in the organic phase O2, the volume ratio of the hydrophobic solvent to the second emulsifier is 100:0.5-5; further 100:1-2;

[0029] In the present invention, the volume ratio of the O1 / W1 type emulsion to the organic phase O2 is 1-2:2-8, and further 1:3-5.

[0030] In the present invention, the chemical formula of the unit aldehyde is R-CHO;

[0031] The R is at least one of a C1-C10 alkyl group, a phenyl group, and a substituted phenyl group;

[0032] In the present invention, the weight ratio of chitosan to unit aldehyde in the O1 / W1 / O2 type emulsion is 25 to 100:1, and can further be 40 to 60:1;

[0033] In the present invention, the temperature of the pre-reaction is 20 to 40°C;

[0034] In the present invention, the pre-reaction time is 0.5 to 4 hours, and can further be 1 to 3 hours;

[0035] In the present invention, the cross-linking agent is a halogenated propylene oxide, preferably epichlorohydrin;

[0036] In the present invention, the volume ratio of chitosan to cross-linking agent in the O1 / W1 / O2 type emulsion is 25 to 100:1, and can further be 40 to 60:1;

[0037] In the present invention, the temperature of the cross-linking reaction is 50 to 80°C;

[0038] Preferably, an acid-binding agent is added during the cross-linking reaction stage, which may be a base or an amine component. Furthermore, the pH of the cross-linking reaction stage is 8 to 10;

[0039] In the present invention, the cross-linking reaction time is 4 to 8 hours.

[0040] In the present invention, after the cross-linking reaction, the cross-linked chitosan is obtained by solid-liquid separation and then washing.

[0041] In the present invention, the cross-linked chitosan is subjected to a two-stage treatment of first acid and then alkali, which can optimize the physicochemical structure of chitosan, improve the exposure of its surface active sites, and thus make it compatible with the immobilization application requirements of the enzyme, thereby achieving the effect of enhancing the enzyme immobilization rate, immobilization stability and immobilized enzyme activity stability.

[0042] In the present invention, the acid solution in the cross-linked chitosan acid treatment stage includes an aqueous solution of an inorganic acid or an organic acid;

[0043] In the present invention, the solute concentration of the acid solution is 0.1 to 2M, and further 0.4 to 1M.

[0044] In the present invention, the temperature and time of the acid treatment can be adjusted as required, for example, the temperature can be 30 to 60° C., further 45 to 55° C. The time of the acid treatment is more than 5 hours, further 8 to 15 hours.

[0045] In the present invention, the alkali solution of the material in the alkali treatment stage is an aqueous solution containing at least one of alkali metal hydroxides, carbonates, and bicarbonates. It is further an aqueous solution of at least one of sodium carbonate and sodium bicarbonate. Studies have shown that the use of preferred carbonates and / or bicarbonates for the treatment can further optimize the physicochemical structure of the modified chitosan, further enhance the activity of the immobilized enzyme of the material, reduce the problem of immobilized enzyme inactivation, and in addition, improve its pH resistance and high temperature stability.

[0046] In the present invention, the solute concentration of the alkali solution is 0.1 to 2M, and further 0.4 to 1M.

[0047] In the present invention, the temperature and time of the alkali treatment can be adjusted as required, for example, the temperature can be 30 to 60° C., further 45 to 55° C. The time of the alkali treatment is more than 0.5 h, further 1 to 4 h.

[0048] In the present invention, the structure of the dialdehyde component is OHC-X-CHO; X is at least one of a carbon chain, a saturated carbon ring, and an aromatic ring;

[0049] In the present invention, the weight ratio of the dialdehyde component to chitosan is 0.5 to 5:100, further 1 to 2:100.

[0050] The invention also provides chitosan microspheres prepared by the preparation method.

[0051] The preparation method of the present invention can give the prepared chitosan microspheres special microscopic characteristics, and the chitosan microspheres with the characteristics prepared by the preparation method can adapt to the enzyme immobilization requirements and improve the enzyme immobilization rate, immobilization stability and immobilized enzyme activity.

[0052] The present invention also provides an application of the chitosan microspheres obtained by the preparation method, using the chitosan microspheres as carriers and compounding with active ingredients to obtain composite materials loaded with active ingredients;

[0053] Preferably, the active ingredient comprises at least one of a pharmaceutical active ingredient and an enzyme;

[0054] Preferably, in the composite material, the compounding amount of the active ingredient is greater than 5 wt.%, further can be greater than 50 wt.%, further can be greater than 80 wt.%.

[0055] The present invention also provides an immobilized enzyme, which comprises chitosan microspheres prepared by the preparation method and an enzyme immobilized thereon;

[0056] In the present invention, the immobilization amount of the enzyme is 5 wt.% or more, further can be 50 wt.% or more, further can be 80 wt.% or more.

[0057] The invention also provides a method for preparing the immobilized enzyme, which comprises mixing the enzyme buffer and the chitosan microspheres, and then performing solid-liquid separation to obtain the immobilized enzyme.

[0058] Beneficial Effects

[0059] The chitosan microspheres prepared by the present invention have good mass transfer performance and adsorption performance, which are beneficial to the adsorption and loading of proteins; the immobilized enzyme prepared by using the immobilized carrier has good protein stability and thermal stability, which is more beneficial to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The relative enzyme activity of the free enzyme and the immobilized enzyme at different temperatures in Example 1; Figure 2 The relative enzyme activity of the free enzyme and the immobilized enzyme at different pH values ​​in Example 1; DETAILED DESCRIPTION

[0060] In order to better illustrate the present invention, the contents of the present invention are explained below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0061] An exemplary method for preparing chitosan microspheres of the present invention includes the following steps:

[0062] (1) dissolving chitosan powder in glacial acetic acid solution, stirring thoroughly to make it completely dissolved, and adding tween 80 and porogen to prepare O1 / W1 type emulsion, which is used as the continuous phase;

[0063] (2) mixing liquid paraffin and span 80 to form a dispersed phase;

[0064] (3) slowly adding the continuous phase dropwise to the dispersed phase and dispersing the phase uniformly to form an O1 / W1 / O2 type emulsion;

[0065] (4) protecting the amino group with benzaldehyde, and then using epichlorohydrin as a cross-linking agent to react to prepare a chitosan carrier;

[0066] (5) washing the carrier with petroleum ether, anhydrous ethanol and pure water in sequence;

[0067] (6) using acid to deprotect the amino group, adding sodium carbonate solution to perform secondary pore making on the carrier, and activating the carrier with glutaraldehyde to obtain an activated chitosan carrier;

[0068] 2. Enzyme immobilization, the method is as follows:

[0069] The present invention also applies it to the preparation of immobilized enzyme, comprising the following steps:

[0070] (1) The enzyme protein is dissolved in a phosphate buffer solution at pH 7.0 to 8.0, the activated chitosan microspheres according to claim (1) are added thereto, and immobilized at 18 to 25° C. for 12 to 48 hours.

[0071] (2) After sufficient immobilization, the supernatant is removed, the filter residue is fully washed with deionized water, and then drained to obtain the immobilized enzyme.

[0072] The following is a specific plan:

[0073] Part I: Preparation of chitosan microspheres

[0074] Example 1

[0075] Step (1):

[0076] Weigh 3 g of chitosan powder and dissolve it in 2% glacial acetic acid solution. Stir thoroughly to dissolve it completely to obtain 100 mL of chitosan solution. Add 4 mL of tween 80 and 15 mL of cyclohexane to prepare an O / W emulsion as the continuous phase.

[0077] Step (2):

[0078] 2 mL of span80 was added to 100 mL of liquid paraffin as the dispersed phase. 连续相 :V 分散相 =1:4, the two were mixed at a stirring speed of 200 rpm, and the mixture was stirred for 1 h to obtain an O / W / O emulsion.

[0079] Step (3):

[0080] Add benzaldehyde (the weight ratio of chitosan to benzaldehyde in the O / W / O emulsion is 50:1) and stir at 200 rpm at 30°C for 2 h. Raise the temperature to 60°C, add epichlorohydrin (the weight ratio of chitosan to epichlorohydrin in the O / W / O emulsion is 50:1), slowly add NaOH solution, and control the pH

[0081] 8-10, 200 rpm stirring for 6 hours, after sufficient reaction, the chitosan carrier is washed with petroleum ether, anhydrous ethanol and deionized water in sequence to obtain a carrier.

[0082] Step (4):

[0083] The washed support was treated with 0.5 M hydrochloric acid at 50 °C for 12 h and then continued in 0.5 M Na 2 CO 3 (alkaline solution) for 2 hours, glutaraldehyde (concentration of 50%, weight ratio of glutaraldehyde to carrier is 1:100) is added for activation at room temperature, and after 2 hours of treatment, the activated carrier is washed with deionized water.

[0084] Example 2

[0085] Compared with Example 1, the only difference is that the conditions of the continuous phase and the dispersed phase in steps (1) to (2) are changed, wherein the difference in step (1) is:

[0086] Weigh 3 g of chitosan powder and dissolve it in 2% glacial acetic acid solution. Stir thoroughly to dissolve it completely to obtain 100 mL of chitosan solution. Add 2 mL of tween 80 and 20 mL of cyclohexane to prepare an O / W emulsion as the continuous phase.

[0087] Step (2):

[0088] Add 1 mL of span80 to 100 mL of liquid paraffin as the dispersed phase. 连续相 :V 分散相 =1:4, the two were mixed at a stirring speed of 200 rpm, and the mixture was stirred for 1 h to obtain an O / W / O emulsion.

[0089] Other operations and parameters are the same as in Example 1.

[0090] Example 3

[0091] Compared with Example 1, the only difference is that the alkali solution of the alkali treatment in step (4) is changed. The experimental groups are:

[0092] Group A: The base is sodium hydroxide.

[0093] Group B: The base is sodium bicarbonate.

[0094] Comparative Example 1

[0095] Compared with Example 1, the only difference is that in step 1, the tween 80 and cyclohexane are not added, and the chitosan solution is directly used as the continuous phase to participate in the treatment of step 2 and subsequent steps, that is, the chitosan is not pre-formed into an O / W emulsion, and the other operations and parameters are the same as in Example 1.

[0096] Comparative Example 2

[0097] Compared with Comparative Example 1, the only difference is that the tween 80 and cyclohexane omitted in step 1 are added to the dispersed phase in equal amounts to directly form a W / O emulsion. Other operations and parameters are the same as in Example 1.

[0098] Comparative Example 3

[0099] Compared with Example 1, the only difference is that cyclohexane is not added in step 1. Other operations and parameters are the same as in Example 1.

[0100] Comparative Example 4

[0101] Compared with Example 1, the only difference is that no alkali treatment is performed in step 4. Other operations and parameters are the same as in Example 1.

[0102] Comparative Example 5

[0103] Compared with Example 1, the only difference is that no acid treatment is performed in step 4. Other operations and parameters are the same as in Example 1.

[0104] Part II: Enzyme Immobilization Applications

[0105] Example 4. Immobilization of deacetylase using chitosan microspheres

[0106] The deacetylase was immobilized using the chitosan microspheres prepared in each case. A certain weight of chitosan microsphere carrier was weighed, and the carrier was added to the enzyme protein-phosphate solution at an enzyme dosage of 1000U / g, and adsorbed at 20°C and 200rpm for 12 hours. The immobilized enzyme was washed with pure water to obtain the deacetylase immobilized enzyme. Among them, the deacetylase loaded on the chitosan microsphere carrier prepared in Example 1 is marked as the fixed deacetylase in Example 1; the deacetylase loaded on the chitosan microsphere carrier prepared in Example 2 is marked as the fixed deacetylase in Example 2; the deacetylase loaded on the chitosan microsphere carrier prepared in Example 3A is marked as the fixed deacetylase in Example 3A; the deacetylase loaded on the chitosan microsphere carrier prepared in Example 3B is marked as the fixed deacetylase in Example 3B; the chitosan microsphere carrier prepared in Comparative Example 1 is loaded The deacetylase loaded on the chitosan microsphere carrier is marked as the fixed deacetylase of Comparative Example 1; the deacetylase loaded on the chitosan microsphere carrier prepared in Comparative Example 2 is marked as the fixed deacetylase of Comparative Example 2; the deacetylase loaded on the chitosan microsphere carrier prepared in Comparative Example 3 is marked as the fixed deacetylase of Comparative Example 3; the deacetylase loaded on the chitosan microsphere carrier prepared in Comparative Example 4 is marked as the fixed deacetylase of Comparative Example 4; the deacetylase loaded on the chitosan microsphere carrier prepared in Comparative Example 5 is marked as the fixed deacetylase of Comparative Example 5.

[0107] Deacetylase immobilized enzyme activity detection method: accurately weigh 0.10g of immobilized enzyme sample, add it to a reactor containing 50mL of substrate solution, keep the temperature at 30℃ in a water bath, adjust the pH to 8.00 with 0.1mol / L sodium hydroxide titration solution, keep the pH at 8.00 for 3 minutes, and record the consumption of sodium hydroxide titration solution.

[0108] Enzyme activity unit: Under certain reaction conditions, the unit amount of enzyme consumes 1 μmol of NaOH per minute.

[0109] Calculation of loading rate: The activity of the enzyme solution before carrier immobilization is measured, which is the original enzyme activity; the activity of the enzyme solution after carrier immobilization is measured, which is the residual activity. Loading rate = (original enzyme activity - residual activity) / original enzyme activity. The calculated loading rate results are shown in Table 1.

[0110] Comparative Example 6. Immobilization of deacetylase using commercial carrier ECHA

[0111] The difference from Example 4 is that the chitosan carrier is replaced by the commercial carrier ECHA purchased by our company, and the other immobilization conditions are the same. The activity of the immobilized enzyme is detected by the enzyme activity detection method in Example 4 and the loading rate is calculated by the formula in Example 4. The results are shown in Table 1.

[0112] Table 1

[0113] name Enzyme activity (U / g) Load rate (%) Example 1 Immobilized deacetylase 222.94 93.23 Example 2 Immobilized deacetylase 220.36 92.46 Example 3A Immobilized deacetylase 201.26 90.3 Example 3B Immobilized deacetylase 218.23 93.1 Comparative Example 1 Immobilized deacetylase 40.89 25.39 Comparative Example 2 Immobilized deacetylase 55.68 28.33 Comparative Example 3 Immobilized deacetylase 41.64 27.65 Comparative Example 4 Immobilized deacetylase 150.42 72.95 Comparative Example 5 Immobilized deacetylase 5.41 3.2 Comparative Example 6 Immobilized deacetylase 120.54 65.54

[0114] (1) Effect of temperature on free enzyme and immobilized enzyme

[0115] The enzyme was immobilized according to the above immobilization method. The immobilized enzyme and free enzyme prepared by the carrier of Example 1 were shaken at 20°C, 25°C, 30°C, 35°C, 40°C, 45°C and 50°C for 60 min, and the enzyme activities of the immobilized enzyme and free enzyme at different temperatures were measured. The highest value of the detected enzyme activity was 100%, and the relative enzyme activities under different temperature conditions were calculated. The results are shown in Figure 1 .

[0116] (2) Effect of pH on free enzyme and immobilized enzyme

[0117] The enzyme was immobilized according to the above immobilization method. The immobilized enzyme and free enzyme prepared by the carrier of Example 1 were oscillated for 60 min at pH 4.0, pH 5.0, pH 6.0, pH 7.0, pH 8.0, pH 9.0 and pH 10.0, respectively, and the enzyme activities of the immobilized enzyme and free enzyme at different temperatures were measured. The highest value of the detected enzyme activity was 100%, and the relative enzyme activities under different temperature conditions were calculated. The results are shown in Figure 2 .

[0118] Example 5. Immobilization of formate dehydrogenase using chitosan microspheres

[0119] The chitosan microsphere carrier of Example 1 was weighed and added to the enzyme protein-phosphate solution at an enzyme dosage of 100 U / g, and adsorbed for 12 hours at 20° C. and 200 rpm. The immobilized enzyme was washed with pure water to obtain the immobilized formate dehydrogenase enzyme.

[0120] Formate dehydrogenase immobilized enzyme activity detection method: The reaction system for enzyme activity determination is 100mL, including 6mmol / L sodium formate, 200mmol / L PBS buffer (pH7.5), 10mmol / L NAD + and 0.10 g of immobilized enzyme, and the absorbance change within 1 min was measured at a wavelength of 340 nm.

[0121] Enzyme activity unit: Under certain reaction conditions, the amount of enzyme that generates or consumes 1 μmol of NADH per minute is one unit.

[0122] Loading ratio: Calculate the loading ratio using the formula in Example 4.

[0123] The activity of the prepared immobilized enzyme can reach 16.4 U / g, and the loading rate is 80.65%.

[0124] Example 6. Immobilization of glutathione synthetase using chitosan microspheres

[0125] Chitosan microspheres were prepared in the same manner as in Example 1, and glutathione synthetase was immobilized using the prepared chitosan microspheres. A certain weight of chitosan microsphere carrier was weighed, and the carrier was added to the enzyme protein-phosphate solution at an enzyme dosage of 400 U / g, and adsorbed at 25° C. and 200 rpm for 24 h. The immobilized enzyme was washed with pure water to obtain an immobilized glutathione synthetase enzyme.

[0126] Method for detecting the activity of immobilized glutathione synthetase: accurately weigh 0.10 g of immobilized enzyme, add it to 20 mL of substrate solution, stir and react in a water bath at 37° C. for 10 min, and perform HPLC analysis.

[0127] Enzyme activity unit: Under certain reaction conditions, the amount of enzyme that produces 1 μmol of glutathione per minute is one unit.

[0128] Loading ratio: Calculate the loading ratio using the formula in Example 4.

[0129] The activity of the prepared immobilized enzyme can reach 64.08 U / g, and the loading rate is 96.84%.

Claims

1. A method for preparing chitosan microspheres, characterized in that the steps include: Step (1): Emulsion preparation Obtaining an aqueous phase W1 in which chitosan is dissolved, and then adding an organic phase O1 containing a first emulsifier and a porogen to the aqueous phase W1 to form an O1 / W1 type emulsion; the porogen is a hydrophobic component; Obtaining an organic phase O2 in which a hydrophobic solvent and a second emulsifier are dissolved, and adding the O1 / W1 type emulsion of step (1) to the organic phase O2 to form an O1 / W1 / O2 type emulsion; Step (2): Emulsion cross-linking The O1 / W1 / O2 type emulsion and the unit aldehyde are pre-reacted, and then mixed with a cross-linking agent for cross-linking to obtain cross-linked chitosan; Step (3): Modification The cross-linked chitosan is treated with acid in advance, then treated with alkali, and then activated with dialdehyde components to obtain the chitosan microspheres.

2. The method for preparing chitosan microspheres according to claim 1, characterized in that: The aqueous phase W1 is an acid aqueous solution containing chitosan; further, an acetic acid aqueous solution containing chitosan; Preferably, in the aqueous phase W1, the concentration of chitosan is 0.01-0.1 g / mL; Preferably, the first emulsifier includes at least one of tween 80 and tween 60; Preferably, the porogen includes at least one of hydrophobic hydrocarbons and esters, and further includes at least one of alkanes, cycloalkanes, olefins, aromatic hydrocarbons, and ethyl acetate having a carbon number within 10; Preferably, in the organic phase O1, the volume ratio of the first emulsifier to the porogen is 1:1-15; further, it can be 1:3-10; Preferably, the volume ratio of the aqueous phase W1 to the organic phase O1 is 100:15-25.

3. The method for preparing chitosan microspheres according to claim 1, characterized in that: The second emulsifier includes at least one of span 80, span 60 and AEO-3; Preferably, in the organic phase O2, the volume ratio of the hydrophobic solvent to the second emulsifier is 100:0.5-5; Preferably, the volume ratio of the O1 / W1 type emulsion to the organic phase O2 is 1-2:2-8.

4. The method for preparing chitosan microspheres according to claim 1, characterized in that: The chemical formula of the unit aldehyde is R-CHO; The R is at least one of a C1-C10 alkyl group, a phenyl group, and a substituted phenyl group; Preferably, the weight ratio of chitosan to unit aldehyde in the O1 / W1 / O2 type emulsion is 25 to 100:1; Preferably, the temperature of the pre-reaction is 20 to 40°C; Preferably, the pre-reaction time is 0.5 to 4 hours; Preferably, the cross-linking agent is a halogenated propylene oxide, preferably epichlorohydrin; Preferably, the volume ratio of chitosan to cross-linking agent in the O1 / W1 / O2 type emulsion is 25 to 100:1; Preferably, the temperature of the cross-linking reaction is 50 to 80°C; Preferably, the pH of the cross-linking reaction is 8 to 10; Preferably, the cross-linking reaction time is 4 to 8 hours.

5. The method for preparing chitosan microspheres according to claim 1, characterized in that: After the cross-linking reaction, solid-liquid separation is performed, followed by washing treatment to obtain the cross-linked chitosan; Preferably, the acid solution in the cross-linked chitosan acid treatment stage comprises an aqueous solution of an inorganic acid or an organic acid; Preferably, the solute concentration of the acid solution is 0.1 to 2 M, further 0.4 to 1 M; Preferably, the alkali solution of the alkali treatment stage material is an aqueous solution containing at least one of alkali metal hydroxide, carbonate, and bicarbonate; Preferably, the solute concentration of the alkali solution is 0.1-2M, further 0.4-1M.

6. The method for preparing chitosan microspheres according to claim 1, characterized in that: The dialdehyde component has a structure of OHC-X-CHO; X is at least one of a carbon chain, a saturated carbon ring, and an aromatic ring; Preferably, the weight-to-volume ratio of the chitosan and dialdehyde components is 100:0.5-5, further 100:1-2.

7. Chitosan microspheres prepared by the preparation method according to any one of claims 1 to 6.

8. An application of chitosan microspheres prepared by the preparation method according to any one of claims 1 to 6, characterized in that: Using it as a carrier and compounding it with active ingredients to obtain a composite material loaded with active ingredients; Preferably, the active ingredient comprises at least one of a pharmaceutical active ingredient and an enzyme; Preferably, in the composite material, the compounding amount of the active ingredient is above 5 wt.%.

9. An immobilized enzyme, characterized in that Comprising chitosan microspheres prepared by the preparation method according to any one of claims 1 to 6, and enzymes immobilized thereon; Preferably, the enzyme immobilization amount is above 5 wt.%.

10. A method for preparing an immobilized enzyme, characterized in that: The enzyme buffer and the chitosan microspheres are mixed, followed by solid-liquid separation to obtain the product.

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