阅读说明：本技术 一种壳聚糖固定化酶及其制备方法 (Chitosan immobilized enzyme and preparation method thereof ) 是由 梁力曼 牛奎 张建平 田宏燕 沈莉 于 2021-11-03 设计创作，主要内容包括：本发明公开了一种壳聚糖固定化酶的制备方法,其包括以下步骤：①配置浓度为1.5mg/ml的壳聚糖溶液、浓度为2.25-2.5mg/ml的酶溶液,②将酶溶液加入壳聚糖溶液中形成预混液,并加入乙醇和异丁醇的混合溶液,于35-40℃下搅拌30min-1h,形成壳聚糖固定化酶的凝胶体,③配置浓度为3-5mg/ml的葡萄糖溶液,凝胶体中加入葡萄糖溶液,于35℃交联4h,④降温冷却,用无水乙醇洗涤,干燥后制得壳聚糖固定化酶颗粒。本发明具有生产时间短、制得的固定化酶酶活力回收率高等优点。(The invention discloses a preparation method of chitosan immobilized enzyme, which comprises the following steps: preparing a chitosan solution with the concentration of 1.5mg/ml and an enzyme solution with the concentration of 2.25-2.5mg/ml, adding the enzyme solution into the chitosan solution to form a premixed solution, adding a mixed solution of ethanol and isobutanol, stirring for 30min-1h at the temperature of 35-40 ℃ to form a gel of the chitosan immobilized enzyme, preparing a glucose solution with the concentration of 3-5mg/ml, adding the glucose solution into the gel, crosslinking for 4h at the temperature of 35 ℃, cooling, washing with absolute ethyl alcohol, and drying to obtain the chitosan immobilized enzyme particles. The invention has the advantages of short production time, high recovery rate of enzyme activity of the prepared immobilized enzyme and the like.)

1. A preparation method of chitosan immobilized enzyme is characterized by comprising the following steps: dissolving chitosan powder in 2% acetic acid solution to prepare 1.5mg/ml chitosan solution, dissolving free enzyme in phosphate buffer solution with pH of 4-6 to prepare 2.25-2.5mg/ml enzyme solution, adding the enzyme solution into the chitosan solution to form premixed solution, adding mixed solution of ethanol and isobutanol, mixing the ethanol and the isobutanol according to the weight ratio of 1:1-1:4, stirring the premixed solution and the mixed solution at the volume ratio of 0.04-0.06:1 for 30min-1h at the temperature of 35-40 ℃ at the stirring speed of 150-200r/min to form chitosan immobilized enzyme gel, dissolving oxidized glucose in absolute ethanol to prepare 3-5mg/ml glucose solution, adding the glucose solution into the gel, crosslinking at the temperature of 35 ℃ for 4h, cooling, washing with absolute ethyl alcohol for 3 times, and drying to obtain the chitosan immobilized enzyme particles.

2. The method for preparing chitosan immobilized enzyme according to claim 1, characterized in that: the concentration of the enzyme solution is 2.35-2.5 mg/ml.

3. The method for preparing chitosan immobilized enzyme according to claim 1, characterized in that: the volume ratio of ethanol to isobutanol in the mixed solution is 1: 3.

4. The method for preparing chitosan immobilized enzyme according to claim 1, characterized in that: the free enzyme is papain.

5. A chitosan immobilized enzyme prepared by the method of any one of claims 1 to 4.

Technical Field

The invention relates to the technical field of immobilized enzymes, in particular to a chitosan immobilized enzyme and preparation thereof.

Background

An immobilized enzyme is an enzyme which has a catalytic action in a certain spatial range and can be repeatedly and continuously used. Generally, enzymatic reactions are carried out in an aqueous solution, and an immobilized enzyme is obtained by physically or chemically treating a water-soluble enzyme to render it insoluble in water but still enzymatically active.

As disclosed in "a device for immobilized enzyme preparation reaction and its application" of chinese patent publication No. CN105316232A, the most commonly used preparation methods of immobilized enzymes at present are cross-linking method and embedding method.

The crosslinking method refers to an immobilization method in which amino acid residues of free enzymes are reacted with a bifunctional or polyfunctional crosslinking agent with the aid of a polyfunctional reagent to cause crosslinking. The commonly used bifunctional reagent comprises glutaraldehyde, diazobenzidine-2-2' -disulfonic acid and the like, and the commonly used multifunctional reagent is toluene-2-isocyano-4-isothiocyanic acid, wherein the glutaraldehyde is most widely applied. Glutaraldehyde has two aldehyde groups, both of which can react with free amino groups of enzyme or protein to form Schiff base, so that enzyme molecules are crosslinked to prepare the immobilized enzyme.

The embedding method is a method of embedding an enzyme in a porous carrier to immobilize the enzyme, and the preparation process is simple and convenient and the conditions are mild. The carrier does not react with the amino acid residue of the enzyme protein, the high-level structure of the enzyme is rarely changed, and higher enzyme activity recovery rate can be obtained. Therefore, the immobilized enzyme prepared by the embedding method has a great advantage relatively. However, carriers commonly used for preparing immobilized enzymes by an embedding method, such as gelatin, agar, agarose, chitosan, sodium alginate and the like, have the characteristic of high viscosity, so that the preparation process has the technical problems of difficult molding and low production efficiency, and large-scale industrial production cannot be realized.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a preparation method of chitosan immobilized enzyme, which has the advantages of short production time, high recovery rate of enzyme activity of the prepared immobilized enzyme and the like.

In order to achieve the purpose, the invention adopts the technical scheme that: a preparation method of chitosan immobilized enzyme is characterized by comprising the following steps: dissolving chitosan powder in 2% acetic acid solution to prepare 1.5mg/ml chitosan solution, dissolving free enzyme in phosphate buffer solution with pH of 4-6 to prepare 2.25-2.5mg/ml enzyme solution, adding the enzyme solution into the chitosan solution to form premixed solution, adding mixed solution of ethanol and isobutanol, mixing the ethanol and the isobutanol according to the weight ratio of 1:1-1:4, stirring the premixed solution and the mixed solution at the volume ratio of 0.04-0.06:1 for 30min-1h at the temperature of 35-40 ℃ at the stirring speed of 150-200r/min to form chitosan immobilized enzyme gel, dissolving oxidized glucose in absolute ethanol to prepare 3-5mg/ml glucose solution, adding the glucose solution into the gel, crosslinking at the temperature of 35 ℃ for 4h, cooling, washing with absolute ethyl alcohol for 3 times, and drying to obtain the chitosan immobilized enzyme particles.

Further, the concentration of the enzyme solution is 2.35-2.5 mg/ml.

Further, the volume ratio of ethanol to isobutanol in the mixed solution is 1: 3.

Further, the free enzyme is papain.

The invention also provides a chitosan immobilized enzyme which is prepared by adopting the method.

By adopting the scheme, the chitosan immobilized enzyme prepared by the invention has the advantages of easy molding, more pores, high enzyme activity recovery rate, short production time and the like.

The invention is further described below with reference to specific embodiments.

Detailed Description

Firstly, the chitosan immobilized enzyme of the specific embodiment of the invention is prepared by the following steps:

dissolving chitosan powder in 2% acetic acid solution to prepare 1.5mg/ml chitosan solution, dissolving papain in 4-6 pH phosphoric acid buffer solution to prepare 2.25-2.5mg/ml enzyme solution,

adding an enzyme solution into the chitosan solution to form a premixed solution, adding the enzyme into a mixed solution of ethanol and isobutanol, mixing the ethanol and the isobutanol according to a weight ratio of 1:1-1:4, wherein the volume ratio of the mixed solution to the premixed solution is 0.04-0.06:1, stirring for 30min-1h at 35-40 ℃ at the stirring speed of 150-,

dissolving the oxidized glucose in absolute ethyl alcohol to prepare a glucose solution with the concentration of 3-5mg/ml, adding the glucose solution into the gel for crosslinking for 4-6h, cooling, washing for 3 times by using the absolute ethyl alcohol, and drying to obtain the chitosan immobilized enzyme particles.

Example 1

Preparing 1.5mg/ml chitosan solution, 2.25mg/ml papain solution, preparing mixed solution of ethanol and isobutanol according to the weight ratio of 1:3,

② mixing 100ml of enzyme solution with 100ml of chitosan solution, adding 12ml of mixed solution of ethanol and isobutanol, stirring for 30min-1h at 35 ℃, the stirring speed is 150-,

dissolving the oxidized glucose in absolute ethyl alcohol to prepare a glucose solution with the concentration of 3mg/ml, adding 1ml of the glucose solution into the gel, dripping the glucose solution into the gel for crosslinking for 4 hours,

cooling, washing with absolute ethyl alcohol, and drying to obtain the chitosan immobilized enzyme particles.

Example 2

Preparing 1.5mg/ml chitosan solution, 2.35mg/ml papain solution, preparing mixed solution of ethanol and isobutanol according to the weight ratio of 1:3,

② mixing 100ml of enzyme solution with 100ml of chitosan solution, adding 12ml of mixed solution of ethanol and isobutanol, stirring for 30min-1h at 35 ℃, the stirring speed is 150-,

dissolving the oxidized glucose in absolute ethyl alcohol to prepare a glucose solution with the concentration of 3mg/ml, adding 1ml of the glucose solution into the gel, dripping the glucose solution into the gel for crosslinking for 4 hours,

cooling, washing with absolute ethyl alcohol, and drying to obtain the chitosan immobilized enzyme particles.

Example 3

Preparing 1.5mg/ml chitosan solution, 2.5mg/ml papain solution, preparing mixed solution of ethanol and isobutanol according to the weight ratio of 1:3,

② mixing 100ml of enzyme solution with 100ml of chitosan solution, adding 11ml of mixed solution of ethanol and isobutanol, stirring for 30min-1h at 35 ℃, the stirring speed is 150-,

dissolving the oxidized glucose in absolute ethyl alcohol to prepare a glucose solution with the concentration of 3mg/ml, adding 1ml of the glucose solution into the gel, dripping the glucose solution into the gel for crosslinking for 4 hours,

cooling, washing with absolute ethyl alcohol, and drying to obtain the chitosan immobilized enzyme particles.

Example 4

Preparing 1.5mg/ml chitosan solution, 2.5mg/ml papain solution, preparing mixed solution of ethanol and isobutanol according to the weight ratio of 1:1,

② mixing 100ml of enzyme solution with 100ml of chitosan solution, adding 8ml of mixed solution of ethanol and isobutanol, stirring for 30min-1h at 35 ℃, the stirring speed is 150-,

dissolving the oxidized glucose in absolute ethyl alcohol to prepare a glucose solution with the concentration of 3mg/ml, adding 1ml of the glucose solution into the gel, dripping the glucose solution into the gel for crosslinking for 4 hours,

cooling, washing with absolute ethyl alcohol, and drying to obtain the chitosan immobilized enzyme particles.

Example 5

Preparing 1.5mg/ml chitosan solution, 2.5mg/ml papain solution, preparing mixed solution of ethanol and isobutanol according to the weight ratio of 1:2,

② mixing 100ml of enzyme solution with 100ml of chitosan solution, adding 9ml of mixed solution of ethanol and isobutanol, stirring for 30min-1h at 35 ℃, the stirring speed is 150-,

dissolving the oxidized glucose in absolute ethyl alcohol to prepare a glucose solution with the concentration of 3mg/ml, adding 1ml of the glucose solution into the gel, dripping the glucose solution into the gel for crosslinking for 4 hours,

cooling, washing with absolute ethyl alcohol, and drying to obtain the chitosan immobilized enzyme particles.

Example 6

Preparing 1.5mg/ml chitosan solution, 2.5mg/ml papain solution, preparing mixed solution of ethanol and isobutanol according to the weight ratio of 1:4,

② mixing 100ml of enzyme solution with 100ml of chitosan solution, adding 12ml of mixed solution of ethanol and isobutanol, stirring for 30min-1h at 35 ℃, the stirring speed is 150-,

dissolving the oxidized glucose in absolute ethyl alcohol to prepare a glucose solution with the concentration of 3mg/ml, adding 1ml of the glucose solution into the gel, dripping the glucose solution into the gel for crosslinking for 4 hours,

cooling, washing with absolute ethyl alcohol, and drying to obtain the chitosan immobilized enzyme particles.

Example 7

Preparing 1.5mg/ml chitosan solution, 2.5mg/ml papain solution, preparing mixed solution of ethanol and isobutanol according to the weight ratio of 1:3,

② mixing 100ml of enzyme solution with 100ml of chitosan solution, adding 11ml of mixed solution of ethanol and isobutanol, stirring for 30min-1h at 35 ℃, the stirring speed is 150-,

dissolving the oxidized glucose in absolute ethyl alcohol to prepare a glucose solution with the concentration of 4mg/ml, adding 1ml of the glucose solution into the gel, dripping the glucose solution into the gel for crosslinking for 4 hours,

cooling, washing with absolute ethyl alcohol, and drying to obtain the chitosan immobilized enzyme particles.

Example 8

Preparing 1.5mg/ml chitosan solution, 2.5mg/ml papain solution, preparing mixed solution of ethanol and isobutanol according to the weight ratio of 1:3,

② mixing 100ml of enzyme solution with 100ml of chitosan solution, adding 11ml of mixed solution of ethanol and isobutanol, stirring for 30min-1h at 35 ℃, the stirring speed is 150-,

dissolving the oxidized glucose in absolute ethyl alcohol to prepare a glucose solution with the concentration of 5mg/ml, adding 1ml of the glucose solution into the gel, dripping the glucose solution into the gel for crosslinking for 4 hours,

cooling, washing with absolute ethyl alcohol, and drying to obtain the chitosan immobilized enzyme particles.

Secondly, the enzymatic activity of the chitosan immobilized enzyme of examples 1 to 9 was measured.

And (3) measuring by adopting a spectrophotometry method: hydrolyzing a casein substrate by protease under the conditions of certain temperature and pH, adding trichloroacetic acid to stop enzyme reaction, precipitating unhydrolyzed casein, absorbing ultraviolet light by filtrate, measuring by an ultraviolet spectrophotometry, and calculating enzyme activity according to the absorption degree. The method comprises the following specific steps.

1. Enzyme diluent

Weighing L-cysteine hydrochloride (C)₃H₇NO₂S·HCL·H₂O)5.27g, sodium chloride (NaCL)23.4g, water 500ml is added for dissolution, ethylene diamine tetraacetic acid 2.23g is taken and water 200ml is added for dissolution, the two solutions are mixed and mixed, 0.1mol/l sodium hydroxide solution or 0.1mol/l hydrochloric acid solution is used for adjusting the pH value to 5.5, and water is added for dilution to 1000 ml.

2. 0.05mol/l disodium hydrogen phosphate solution

Disodium hydrogen phosphate (Na) was weighed₂HPO₄·12H₂O)17.89g, adding water to dissolve, and fixing the volume to 1000 ml.

3. Casein solution

0.6g (to the nearest 0.0002g) of casein which has been faithfully dried to a constant weight by means of a silica gel dryer is weighed out and placed in a beaker, if 80ml of 0.05mol/l disodium hydrogenphosphate solution is used. The mixture was stirred while heating in a boiling water bath until it was completely dissolved, and after cooling, the mixture was adjusted to pH 7.0 with 0.1mol/l hydrochloric acid, transferred to a 100ml volumetric flask, and water was added to the scale.

4. Trichloroacetic acid solution

Weighing 8.995g of trichloroacetic acid, adding 14.97g of anhydrous sodium acetate and 9.45ml of glacial acetic acid, adding a proper amount of water for dissolving, adding water to 500ml, and shaking uniformly.

5. Tyrosine standard solution

Weighing 50mg (precisely 0.0002g) of tyrosine dried to a balance weight at 105 ℃, dissolving with 0.1mol/l hydrochloric acid, transferring into a 100ml volumetric flask, adjusting to the scale with 0.1mol/l hydrochloric acid, and shaking up to obtain a solution containing 50ug/ml of tyrosine.

6. Sample solution

Weighing 0.9g (accurate 0.0002g) of chitosan immobilized enzyme, placing the chitosan immobilized enzyme in a mortar, adding a small amount of enzyme diluent, grinding for 20min, transferring the enzyme diluent to a volumetric flask of 250ml, adding the enzyme diluent to a scale, and fully and uniformly shaking; taking out 1ml of the above liquid, diluting with enzyme diluent, diluting to a constant volume of 20ml, and shaking thoroughly for testing (within 60 min).

7. Measurement procedure

Sucking 1.00ml of sample solution, placing the sample solution into a 10ml colorimetric tube with a plug, preserving the heat in a water bath at 37 +/-0.2 ℃ for 10min, adding 5.00ml of casein solution preheated in the water bath at 37 +/-0.2 ℃, shaking up, placing the sample solution in the water bath at 37 +/-0.2 ℃, reacting for ten minutes, adding 5.00ml of trichloroacetic acid solution preheated in the water bath at 37 +/-0.2 ℃, shaking up, placing the sample solution in the water bath at 37 +/-0.2 ℃ for 40min, filtering the solution by using dry filter paper, taking filtrate, and measuring the absorbance A1 of the filtrate at 275nm wavelength within 2 hours by using distilled water as a reference.

Blank test: sucking 1.00ml of sample solution, placing the sample solution into a 10ml colorimetric tube with a plug, preserving the heat in a water bath at 37 +/-0.2 ℃ for 10min, adding 5.00ml of trichloroacetic acid solution preheated in the water bath at 37 +/-0.2 ℃, shaking up, reacting for 10min, adding 5.00ml of casein solution preheated in the water bath at 37 +/-0.2 ℃, shaking up, placing the mixture in the water bath at 37 +/-0.2 ℃ for 40min, filtering the mixture by using dry filter paper, taking the filtrate, and taking the absorbance A2 of the filtrate with initial distilled water with the wavelength of 275nm as a reference number in 2 h.

Absorbance of tyrosine standard solution: the absorbance A3 of a 50ug/ml tyrosine standard solution was measured at a wavelength of 275nm using distilled water as a reference.

The results were calculated according to the following formula:

the papain activity X1(U/g) in the sample was calculated according to the formula (1):

(1)

in the formula: a1-absorbance of sample solution;

a2-absorbance of blank test;

a3-absorbance of tyrosine standard solution;

m 1-amount of tyrosine contained per ml in micrograms (μ g) in control solution;

m 2-mass of sample in grams (g);

n is the dilution factor of the sample solution.

U/g represents the activity of the immobilized enzyme, and the recovery rate is calculated according to the following formula:

the immobilized enzyme activity recovery (%): immobilized enzyme activity (U)/total solution enzyme activity (U) × 100%. Wherein, the activity of the immobilized enzyme refers to the activity of the immobilized enzyme measured after the enzyme is immobilized, and the total activity of the solution enzyme refers to the activity of the enzyme in the enzyme solution before immobilization.

The test results are shown in Table 1 below

TABLE 1 recovery of vitality of immobilized enzyme

In the above examples 1 to 8, it is understood that the activity of the immobilized enzyme is 31.00 to 38.22U/g and the recovery rate of the activity of the immobilized enzyme is 56.14 to 66.12%, and in examples 1 to 3, the attachment of the enzyme to the chitosan carrier is affected depending on the concentration of the enzyme solution, whereas in examples 4 to 6, the difference in the ratio and volume of ethanol and isobutanol affects the pore formation on the surface of the chitosan carrier, and when the content of isobutanol is high, the gel formation can be accelerated, but the specific surface area is relatively small, whereas, the gel formation is slow or difficult, the specific surface area is large after the formation, and when the concentration of the glucose solution is high, the mechanical strength of the immobilized enzyme is affected, thereby affecting the recovery rate.

The present invention is not limited to the above embodiments, and those skilled in the art can implement the present invention in other embodiments according to the disclosure of the present invention, or make simple changes or modifications on the design structure and idea of the present invention, and fall into the protection scope of the present invention.