CN100445377C - Bionic preparing process of silica-alginic acid microcapsule for immobilized beta-glucurosidase - Google Patents

Abstract

The invention discloses a method for biomimetic preparation of silicon dioxide-alginic acid microcapsules for immobilizing β-glucuronidase. The process of the method is as follows: mix β-glucuronidase solution, calcium chloride solution and sodium carboxymethyl cellulose solution in a certain proportion and then drop them into sodium alginate solution to obtain alginic acid microcapsules; Immerse in calcium chloride solution for curing; immerse the cured alginic acid microcapsules in protamine sulfate or protamine solution for a period of time to obtain alginic acid microcapsules coated with protamine; The alginic acid microcapsules are immersed in the sodium silicate solution to synthesize the silica shell to obtain the silica-alginic acid microcapsules. The preparation method of the microcapsule provided by the invention is simple and easy, effectively inhibits the swelling of the alginic acid microcapsule, and the obtained immobilized enzyme has high activity and good reusability.

Description

Method for biomimetic preparation of silica-alginic acid microcapsules for immobilization of β-glucuronidase

technical field

The invention relates to a method for biomimetic preparation of silicon dioxide-alginic acid microcapsules for immobilizing β-glucuronidase, which belongs to the enzyme immobilization technology.

Background technique

Alginic acid microcapsules are a commonly used immobilized enzyme carrier. It is composed of an enzyme-containing polymer solution liquid core and an alginic acid capsule wall, which is similar to the existence of free enzymes in nature. Large, good mass transfer performance and other advantages. However, because the alginic acid polymer material used is easy to swell in aqueous solution and has poor mechanical strength, the reusability of this type of immobilized enzyme is greatly limited. Coating the silica shell on the alginic acid capsule wall can effectively solve the swelling problem of the alginic acid microcapsule.

At present, the sol-gel method is widely used to prepare the silica shell by hydrolysis and polycondensation of organosilane precursors. The sol-gel process needs to be carried out under the catalysis of acid or alkali, and alcohol by-products will be produced at the same time. The existence of these substances has a negative impact on the maintenance of biological enzyme activity, resulting in reduced or even inactivated enzyme activity. In addition, the morphology and structure of the silica shell formed by the sol-gel method are difficult to control. Under the preparation conditions, the silica oligomer produced in the gelation process is negatively charged, which is the same as the surface of alginic acid microcapsules, and there is electrostatic repulsion between the two, so the direct deposition on the surface of alginic acid microcapsules The binding effect between the silica shell and the alginic acid capsule wall is not ideal.

Diatoms in nature are single-celled algae plants, whose soft protoplasts are supported and protected by hard siliceous cell walls, and grow into individuals with a certain shape. Studies have shown that this type of diatom plants build cell walls through biosilicification with the participation of organic matter, that is, a certain organic template promotes and regulates the deposition of silica through electrostatic attraction, and the template itself is finally wrapped in the organic template. Inside the formed silica network, an organic-inorganic hybrid structure is formed. Existing biomimetic silicification studies have shown that the use of natural or organically synthesized templates, such as silaffins, polylysine, polyethyleneimine, etc., can regulate the rapid polymerization of silica at room temperature and neutral conditions to form organic compounds. Biomimetic hybrid materials with advantages of materials and inorganic materials.

Contents of the invention

The purpose of the present invention is to provide a method for biomimetic preparation of silica-alginic acid microcapsules for immobilizing β-glucuronidase. The microcapsule prepared by the method is used for immobilizing enzymes, and has high enzyme activity and good reusability.

The present invention is achieved through the following technical scheme, a method for biomimetic preparation of silica-alginic acid microcapsules for immobilizing β-glucuronidase, characterized in that it comprises the following steps:

(1) Preparation of alginic acid microcapsules: beta-glucuronidase solution with concentration of 0.5mg/ml, calcium chloride solution with concentration of 0.6mol/L and carboxymethyl solution with concentration of 3.0%w/v The sodium cellulose solution was mixed evenly at a volume ratio of 10:8:32, added dropwise to 1.0% w/v sodium alginate solution, stirred for 30-60 minutes, diluted with deionized water, filtered, and then immersed in 0.1mol/L alginate solution. Solidify in calcium chloride solution for 10 minutes to obtain alginic acid microcapsules with a diameter of about 2.5-3.5 mm.

(2) Coating of protamine on the outer surface of alginic acid microcapsules: immerse the alginic acid microcapsules obtained in step (1) in 2-20 mg/ml protamine sulfate or protamine solution, soak for 30-120 min After filtration, protamine-coated alginic acid microcapsules are obtained.

(3) Biomimetic synthesis of silica shell under the control of protamine: immerse the microcapsules obtained in step (2) in 30-80 mmol/L sodium silicate solution with a pH of 5-7 for 60-120 min , and filtered to obtain silica-alginic acid microcapsules.

The preparation method proposed by the invention has the advantages of mild preparation conditions, good anti-swelling performance of the obtained capsule, high enzyme activity maintenance rate of the contained β-glucuronidase, and good repeated use stability.

Description of drawings

Fig. 1 is the energy dispersive spectroscopy (EDS) spectrogram of the surface of the silica-alginic acid microcapsule prepared in Example 3.

Fig. 2 is a scanning electron microscope (SEM) photo of the silica-alginic acid microcapsule prepared in Example 3.

3 is a scanning electron microscope (SEM) photo of the surface of the silica-alginic acid microcapsule prepared in Example 3.

Fig. 4 is a scanning electron microscope (SEM) photo of the section of the silica-alginic acid microcapsule prepared in Example 3.

Detailed ways

Embodiment one

Accurately weigh 1.0mg of β-glucuronidase, dissolve it in 30mmol/L Tris-HCl (trishydroxymethylaminomethane-hydrochloric acid) buffer solution, and dilute to 10ml to obtain 0.5mg/ml enzyme solution. Weigh 160mg protamine sulfate, dissolve in 30mmol/L Tris-HCl buffer solution, and set the volume to 80ml to obtain 2mg/ml protamine sulfate solution. Sodium silicate was dissolved in deionized water, and the pH of the solution was adjusted to 7.0 with 2mol/L HCl to prepare a 50mmol/L sodium silicate solution.

Draw 0.40ml of enzyme solution, add 0.32ml of 0.6mol/L calcium chloride solution, then add 1.28ml of 3.0% w/v sodium carboxymethylcellulose solution, mix with a magnetic stirrer until a uniform solution is formed, and then dissolve the solution Inhale into the syringe, drop into 40ml of stirred sodium alginate solution with a concentration of 1.0% w/v through a needle with an inner diameter of 0.45mm, continue stirring for 30min, add 160ml of deionized water to dilute, filter out the microcapsules, and wash with deionized water . Blot the moisture on the surface of the microcapsules with filter paper, add 10 ml of calcium chloride solution with a concentration of 0.1 mol/L, stir magnetically for 10 min, filter, and wash with water.

Dry the moisture on the surface of the microcapsules with filter paper and put it into 35ml of protamine sulfate solution with a concentration of 2mg/ml, let it stand for 60min, filter it out, then put it into 70ml of sodium silicate solution with a concentration of 50mmol/L, let it stand After 120 minutes, filter out and wash with water to obtain silica-alginic acid microcapsules (1). According to EDS analysis, the mass percentage of silicon element on the surface of the microcapsule is 13.6%, and the swelling degree of the microcapsule in water is 55%.

Embodiment two

Accurately weigh 1.0mg of β-glucuronidase, dissolve it in 30mmol/L Tris-HCl buffer solution, and dilute to 10ml to obtain 0.5mg/ml enzyme solution. Weigh 400mg protamine sulfate, dissolve in 30mmol/L Tris-HCl buffer solution, and set the volume to 80ml to obtain 5mg/ml protamine sulfate solution. Dissolve sodium silicate with deionized water, adjust the pH to 7.0 with 2mol/L HCl, and prepare a 50mmol/L sodium silicate solution.

Draw 0.40ml of enzyme solution, add 0.32ml of 0.6mol/L calcium chloride solution, then add 1.28ml of 3.0% w/v sodium carboxymethylcellulose solution, mix with a magnetic stirrer until a uniform solution is formed, and then dissolve the solution Inhale into the syringe, drop into 40ml of stirred sodium alginate solution with a concentration of 1.0% w/v through a needle with an inner diameter of 0.45mm, continue stirring for 30min, add 160ml of deionized water to dilute, filter out the microcapsules, and wash with deionized water . Blot the moisture on the surface of the microcapsules with filter paper, add 10 ml of calcium chloride solution with a concentration of 0.1 mol/L, stir magnetically for 10 min, filter, and wash with water.

Blot the moisture on the surface of the microcapsules with filter paper and put it into 35ml of protamine sulfate solution with a concentration of 5mg/ml, let it stand for 30min, filter it out, then put it into 70ml of a sodium silicate solution with a concentration of 50mmol/L, let it stand for 120min , filtered out, and washed with water to obtain silica-alginic acid microcapsules (2). According to EDS analysis, the mass percentage of silicon element on the surface of the microcapsule is 28.2%, and the swelling degree of the microcapsule in water is 11%.

Embodiment Three

Accurately weigh 1.0mg of β-glucuronidase, dissolve it in 30mmol/L Tris-HCl buffer solution, and dilute to 10ml to obtain 0.5mg/ml enzyme solution. Weigh 400mg protamine sulfate, dissolve in 30mmol/L Tris-HCl buffer solution, and set the volume to 80ml to obtain 5mg/ml protamine sulfate solution. Dissolve sodium silicate with deionized water, adjust the pH to 7.0 with 2mol/L HCl, and prepare a 50mmol/L sodium silicate solution.

Draw 0.40ml of enzyme solution, add 0.32ml of 0.6mol/L calcium chloride solution, then add 1.28ml of 3.0% w/v sodium carboxymethylcellulose solution, mix with a magnetic stirrer until a uniform solution is formed, and then dissolve the solution Inhale into the syringe, drop into 40ml of stirred sodium alginate solution with a concentration of 1.0% w/v through a needle with an inner diameter of 0.45mm, continue stirring for 30min, add 160ml of deionized water to dilute, filter out the microcapsules, and wash with deionized water . Blot the moisture on the surface of the microcapsules with filter paper, add 10 ml of calcium chloride solution with a concentration of 0.1 mol/L, stir magnetically for 10 min, filter, and wash with water.

Blot the moisture on the surface of the capsule with filter paper and put it into 35ml of protamine sulfate solution with a concentration of 5mg/ml, let it stand for 60min, filter it out, then put it into 70ml of sodium silicate solution with a concentration of 50mmol/L, let it stand for 120min, Filter out and wash with water to obtain silica-alginic acid microcapsules (3). According to EDS analysis, the mass percentage of silicon element on the surface of the capsule is 34.2%, and the microcapsule basically does not swell in water.

Embodiment Four

Dissolve baicalin and anhydrous sodium sulfite into a 30mmol/L Tris-HCl buffer solution to form a solution with a baicalin concentration of 0.09mol/L and anhydrous sodium sulfite concentration of 0.1% w/v, and add the solution containing The silicon dioxide-alginic acid microcapsules of β-glucuronidase are used to carry out the conversion reaction of baicalin under stirring conditions at 37°C. Within a certain time interval, 100 μl of the reaction solution is taken out, and the baicalin is determined by high performance liquid chromatography. The production amount of β-glucuronidase was obtained to obtain the enzyme activity of immobilized β-glucuronidase.

Comparative example one

Accurately weigh 1.0mg of β-glucuronidase, dissolve it in 30mmol/L Tris-HCl buffer solution, and dilute to 10ml to obtain 0.5mg/ml enzyme solution. Draw 0.40ml of enzyme solution, add 0.32ml of 0.6mol/L calcium chloride solution, then add 1.28ml of 3.0% w/v sodium carboxymethylcellulose solution, mix with a magnetic stirrer until a uniform solution is formed, and then dissolve the solution Inhale into the syringe, drop into 40ml of stirred sodium alginate solution with a concentration of 1.0% w/v through a needle with an inner diameter of 0.45mm, continue stirring for 30min, add 160ml of deionized water to dilute, filter out the capsule, and wash with deionized water. Blot the moisture on the capsule surface with filter paper, add 10ml of calcium chloride solution with a concentration of 0.1mol/L, stir for 10min with magnetic force, filter and wash with water to obtain alginic acid microcapsules. It was determined that the swelling degree of the microcapsules in water was 114%.

Comparative example two

Dissolve baicalin and anhydrous sodium sulfite in 30mmol/L Tris-HCl buffer solution, make the baicalin concentration be 0.09mol/L, the solution that anhydrous sodium sulfite concentration is 0.1%w/v, add the preparation containing The alginic acid microcapsules of β-glucuronidase, at 37°C, carry out the conversion reaction of baicalin under the condition of stirring, and take out 100 μl of the reaction solution within a certain time interval, and use high performance liquid chromatography to determine the formation of baicalin The amount of enzyme activity of immobilized β-glucuronidase was obtained.

Table 1 shows the enzymatic activity of the baicalin conversion reaction of the microcapsules containing β-glucuronidase as determined in Example 4 and Comparative Example 2 and the number of repeated uses where the enzymatic activity did not decrease.

Table 1

Claims (1)

1. the method for the silicon-dioxide-alginate microcapsule of bionical being used for fixing of a preparation beta-glucuronidase enzyme is characterized in that may further comprise the steps:

(1) preparation of alginate microcapsule: with concentration is the beta-glucuronidase enzyme solution of 0.5mg/ml, to be the calcium chloride solution of 0.6mol/L and carboxymethylcellulose sodium solution that concentration is 3.0%w/v mix by 10: 8: 32 volume ratio concentration, be added drop-wise in the sodium alginate soln of 1.0%w/v, stir 30～60min, deionized water dilution after-filtration, immerse again in the calcium chloride solution of 0.1mol/L and solidify 10min, obtain the alginate microcapsule of diameter 2.5～3.5mm;

(2) protamine is in the coating of alginate microcapsule outside surface: the alginate microcapsule that step (1) is obtained immerses in the protamine sulfate solution of 2mg/ml or 5mg/ml, soaks 30～120min after-filtration, obtains the alginate microcapsule that protamine coats;

(3) protamine regulation and control silica shell down is bionical synthetic: the micro-capsule that step (2) is obtained immerses 30～80mmol/L, and pH in 5～7 the sodium silicate solution, soaks 60～120min, and filtration obtains silicon-dioxide-alginate microcapsule.

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