阅读说明：本技术 一种离子液体再生纤维素固定化酶的制备方法 (Preparation method of ionic liquid regenerated cellulose immobilized enzyme ) 是由 林金新 黄平 郭小雷 于 2020-05-13 设计创作，主要内容包括：本发明涉及酶的固定化技术领域,尤其涉及一种离子液体再生纤维素固定化酶的制备方法。其包括以下步骤：以离子液体作为溶剂、溶解纤维素以制成离子液体再生纤维素微球载体；通过高碘酸钠对微球载体进行改性；通过还原型谷胱甘肽对酶进行激活处理；最后将酶和微球载体混合负载,得到固定化酶。通过本发明的制备方式能够有效提高酶的固定化率和酶活。(The invention relates to the technical field of enzyme immobilization, in particular to a preparation method of an ionic liquid regenerated cellulose immobilized enzyme. Which comprises the following steps: dissolving cellulose by taking ionic liquid as a solvent to prepare an ionic liquid regenerated cellulose microsphere carrier; modifying a microsphere carrier by sodium periodate; activating enzyme by reduced glutathione; and finally, mixing and loading the enzyme and the microsphere carrier to obtain the immobilized enzyme. The preparation method can effectively improve the immobilization rate and the enzyme activity of the enzyme.)

1. A preparation method of ionic liquid regenerated cellulose immobilized enzyme is characterized by comprising the following steps: the method comprises the following steps:

preparing a carrier: dissolving cellulose in ionic liquid to obtain ionic liquid/cellulose mixed solution; then dripping the ionic liquid/cellulose mixed solution into deionized water by using an injector to ensure that the ionic liquid/cellulose mixed solution is regenerated into small balls; washing the microspheres with deionized water, and naturally drying to obtain microsphere carriers;

modification of a carrier: adding the microsphere carrier into a sodium periodate solution, taking out the microsphere carrier after modification, and directly freezing and freeze-drying to obtain the sodium periodate modified microsphere carrier;

enzyme treatment: adding reduced glutathione into the enzyme solution, and enabling the concentration of the reduced glutathione to be 0.3-0.8 mg/mL to obtain enzyme solution after active treatment;

carrying enzyme: adding the modified microsphere carrier into the enzyme solution after the activity treatment, sealing, placing in a water bath at 25-30 ℃, oscillating for 12-16 h, taking out the microsphere carrier, washing with deionized water, freezing and freeze-drying to obtain the immobilized enzyme.

2. The preparation method of the ionic liquid regenerated cellulose immobilized enzyme according to claim 1, which is characterized in that: in the step (2), a sodium periodate solution with the concentration of 0.5-1 mol/L is adopted.

3. The preparation method of the ionic liquid regenerated cellulose immobilized enzyme according to claim 1, which is characterized in that: in the step (2), the adding proportion of the microsphere carrier to the sodium periodate is 1 g: (0.05-0.1) mol.

4. The preparation method of the ionic liquid regenerated cellulose immobilized enzyme according to claim 1, which is characterized in that: in the step (2), the microsphere carrier is added into a sodium periodate solution to react for 90-120 min.

5. The preparation method of the ionic liquid regenerated cellulose immobilized enzyme according to claim 1, which is characterized in that: in the step (3), the concentration of the enzyme solution is 4-7 mg/mL.

6. The preparation method of the ionic liquid regenerated cellulose immobilized enzyme according to claim 1, which is characterized in that: in the step (4), the mixing mass ratio of the enzyme to the microsphere carrier is (0.25-0.3): 1.

7. the preparation method of the ionic liquid regenerated cellulose immobilized enzyme according to claim 1, which is characterized in that: the freeze-drying conditions are as follows: and (3) carrying out freeze drying for 18-24 h at-85 to-75 ℃ by using liquid nitrogen.

8. The preparation method of the ionic liquid regenerated cellulose immobilized enzyme according to claim 1, which is characterized in that: in the step (1), the adding mass ratio of the cellulose to the ionic liquid is (10-15): 100.

9. the preparation method of the ionic liquid regenerated cellulose immobilized enzyme according to claim 1, which is characterized in that: in the step (1), cellulose is dissolved in ionic liquid, the ionic liquid is placed at 100-120 ℃ and is dissolved for 5-7 hours under magnetic stirring, and then the ionic liquid/cellulose mixed solution is obtained after standing for 12-16 hours.

10. The preparation method of the ionic liquid regenerated cellulose immobilized enzyme according to claim 1, which is characterized in that: the ionic liquid is BMIM]Cl、[C₄MIM]One of Cl.

Technical Field

The invention relates to the technical field of enzyme immobilization, in particular to a preparation method of an ionic liquid regenerated cellulose immobilized enzyme.

Background

Gamma-aminobutyric acid (GABA) is a naturally occurring functional amino acid, has the functions of treating hypertension, epilepsy and Parkinson's disease, preventing arteriosclerosis, regulating arrhythmia, easing pain, resisting skin aging and the like, and has very important values in medical treatment and health care. GABA in organisms is mostly produced by the decarboxylation of glutamate catalyzed by glutamate decarboxylase (GAD), the coenzyme of which is pyridoxal 5-phosphate. The GABA preparation method as a principle of medicines or health products mainly comprises 3 methods of chemical synthesis, separation and extraction and biological synthesis, and the biological synthesis method becomes a mainstream technology. The biosynthesis method is to convert glutamic acid into GABA by using immobilized glutamate decarboxylase (IGAD).

The enzyme is immobilized by binding the enzyme on a carrier material, so that the enzyme is separated from the whole body and can still react with a substrate. The carrier material is an important part of the immobilized enzyme, the structure and performance of the carrier material have great influence on various performances of the immobilized enzyme, and most of the commonly used immobilized carriers at present are hydrogel, carbon fiber, cellulose and the like. The cellulose is an organic resource with the widest distribution and the highest content in the nature, and a product prepared from the natural cellulose can be biodegraded and meets the requirements of modern environmental protection, green chemistry and sustainable development. However, cellulose macromolecules are unbranched linear molecules, a large number of hydrogen bonds exist in molecular chains and molecules, and due to the large number of hydrogen bonds and the complex morphological structure of coexistence of a crystalline region and an amorphous region, natural cellulose has the problems of non-melting and non-dissolving of most solvents, and is the biggest limitation of application of the natural cellulose.

Ionic liquids, also known as low temperature molten salts, are in a liquid state at or near room temperature and are salts consisting entirely of anions and cations. The cationic polymer is generally composed of organic cations and inorganic anions, common cations comprise quaternary phosphonium salt ions, quaternary ammonium salt ions, pyrrole salt ions, imidazole salt ions and the like, and anions comprise halogen ions, hexafluorophosphate ions, tetrafluoroborate ions and the like. The ionic liquid has the advantages of no toxicity, good solubility, no volatilization, good thermal stability, wider liquid temperature area, nonflammability, reusability and the like, and can replace the traditional solvent which is easy to volatilize and pollute. The ionic liquid can be used for dissolving cellulose, and provides a basal hall for the immobilization of bioactive macromolecules.

The patent document with the publication number of CN103667243A discloses a method for preparing a cellulase immobilized carrier by straws treated by ionic liquid and a modifier, which comprises the following steps: pretreatment of the straws, modification of the straws and drying and forming of the carrier. The application adopts the cellulose material obtained by modifying straws with the ionic liquid and the modifier to prepare the enzyme immobilized carrier, and has the problems that the enzyme adsorbed on the carrier is easy to fall off, and the stability and the activity of the immobilized enzyme are not high.

Disclosure of Invention

The invention aims to solve the problems and provides a preparation method of an ionic liquid regenerated cellulose immobilized enzyme.

The technical scheme for solving the problems is to provide a preparation method of an ionic liquid regenerated cellulose immobilized enzyme, which comprises the following steps:

(1) preparing a carrier: dissolving cellulose in ionic liquid to obtain ionic liquid/cellulose mixed solution; then dripping the ionic liquid/cellulose mixed solution into deionized water by using an injector to ensure that the ionic liquid/cellulose mixed solution is regenerated into small balls; washing the microspheres with deionized water, and naturally drying to obtain microsphere carriers;

(2) modification of a carrier: adding the microsphere carrier into a sodium periodate solution, taking out the microsphere carrier after modification, and directly freezing and freeze-drying to obtain the sodium periodate modified microsphere carrier;

(3) enzyme treatment: adding reduced glutathione into the enzyme solution, and enabling the concentration of the reduced glutathione to be 0.3-0.8 mg/mL to obtain enzyme solution after active treatment;

(4) carrying enzyme: adding the modified microsphere carrier into the enzyme solution after the activity treatment, sealing, placing in a water bath at 25-30 ℃, oscillating for 12-16 h, taking out the microsphere carrier, washing with deionized water, freezing and freeze-drying to obtain the immobilized enzyme.

In the step (2), sodium periodate is adopted to modify the microsphere carrier, the oxidation effect of the sodium periodate can enable the continuous diol groups of the cellulose to be broken to generate aldehyde groups (-CHO), and the aldehyde groups can be combined with amino groups (-NH) in enzyme molecules₂) And the covalent bond is formed, so that the enzymatic activity of the immobilized enzyme can be improved. Meanwhile, in the step (2), the modified microsphere carrier is not washed by deionized water and is directly freeze-dried, so that part of sodium periodate is attached to the microsphere carrier, and preparation is made for the subsequent steps.

Substances that enhance the activity of enzymes and accelerate the enzymatic reaction are called activators. The activating agent has a plurality of types, including (i) inorganic cations such as sodium ions, potassium ions, copper ions, calcium ions and the like; ② inorganic anions such as chloride ion, bromide ion, iodide ion, sulfate ion phosphate ion and the like; ③ organic compounds, such as vitamin C, cysteine, reductive glutathione, etc. In step (3) of the present application, reduced glutathione is used as an enzyme stimulant, which can stimulate the activity of the enzyme.

In the step (4), a microsphere carrier containing sodium periodate is added into an enzyme solution containing reducing glutathione, and the structure of the reducing glutathione contains an active sulfhydryl-SH, so that the reducing glutathione is easy to oxidize and dehydrogenate; sodium periodate selectively oxidizes sulfides to sulfoxides, which can be oxidized to sulfones at elevated temperatures or with the addition of excess sodium periodate. Therefore, under the selective oxidation action of sodium periodate, sulfydryl is oxidized into sulfoxide group or even sulfone group, the sulfoxide group and the sulfone group have strong electron withdrawing property, cellulose has electron donating group-OH, enzyme has electron donating group-NH₂. Therefore, oxidized glutathione becomes an intermediate connection structure, and is respectively combined with a cellulose electron-donating group and an enzyme electron-donating group through an electron-withdrawing group, so that the enzyme is firmly loaded on the microsphere carrier, and the immobilization stability and the enzyme activity are effectively improved.

Along with the continuous increase of the concentration of sodium periodate, the quantity of glycol groups broken by cellulose is increased, the quantity of aldehyde groups generated is also increased, and the enzyme activity of immobilized enzyme is also improved; however, the higher the concentration of sodium periodate, the better, the higher the concentration of sodium periodate, and the higher the concentration of sodium periodate. Therefore, in the preferred embodiment of the present invention, sodium periodate is used in the step (2) at a concentration of 0.5 to 1 mol/L.

Preferably, in the step (2), the adding ratio of the microsphere carrier to the sodium periodate is 1 g: (0.05-0.1) mol.

Preferably, in the step (2), the microsphere carrier is added into the sodium periodate solution to react for 90-120 min.

The initial enzyme concentration also has a certain influence on the enzyme activity, the larger initial enzyme concentration can increase the steric hindrance of enzyme molecule quality inspection, the competition among enzymes is severe, so that the immobilization rate of the enzyme is less, and the immobilized enzyme activity is reduced, therefore, as the optimization of the invention, in the step (3), the concentration of the enzyme solution is 4-7 mg/mL.

Preferably, in the step (4), the mixing mass ratio of the enzyme to the microsphere carrier is (0.25-0.3): 1.

as preferred in the present invention, the freeze-drying conditions are all: and (3) carrying out freeze drying for 18-24 h at-85 to-75 ℃ by using liquid nitrogen.

Preferably, in the step (1), the adding mass ratio of the cellulose to the ionic liquid is (10-15): 100.

preferably, in the step (1), the cellulose is dissolved in the ionic liquid, the ionic liquid is placed at 100-120 ℃ for magnetic stirring and dissolution for 5-7 h, and then the ionic liquid/cellulose mixed solution is obtained after standing for 12-16 h.

Preferably, the ionic liquid is selected from [ BMIM ] Cl or [ C4MIM ] Cl.

The invention has the beneficial effects that:

1. the application takes the regenerated cellulose pretreated by the ionic liquid as an immobilized enzyme carrier, and has the advantages of easy recovery, easy biodegradation and environmental protection.

2. According to the method, the sodium periodate modified carrier can effectively improve the loading rate of the enzyme loaded on the carrier and the enzyme activity.

3. The enzymatic activity is further improved by stimulating the enzymatic activity by the reductive glutathione.

4. According to the method, the sulfide can be oxidized into sulfoxide through the sodium periodate, an electron-withdrawing group is obtained, and the enzyme is stably loaded on the carrier through the combination with the electron-supplying group, so that the immobilized enzyme is not easy to fall off.

Detailed Description

The following are specific embodiments of the present invention and further describe the technical solutions of the present invention, but the present invention is not limited to these examples.