阅读说明：本技术 β-葡萄糖苷酶固定化酶的制备方法 (Preparation method of beta-glucosidase immobilized enzyme ) 是由 史楠 王海宾 严文霞 王芳 高莉 李紫玉 郭建峰 杨沛阳 于 2019-10-22 设计创作，主要内容包括：本发明公开了一种β-葡萄糖苷酶固定化酶的制备方法,包括以下步骤：将预处理好的大孔树脂置于容器中,向容器中加入PBS缓冲液,平衡后再加入β-葡萄糖苷酶原溶液,然后将容器置于恒温振荡培养箱中振荡吸附,吸附结束后,将大孔树脂与剩余液体分离,用PBS缓冲液将分离后的大孔树脂冲洗至冲洗液中检测不到蛋白,然后再加入PBS缓冲液和戊二醛试剂进行交联,交联结束后,用PBS缓冲液清洗大孔树脂数次,减压干燥后即得β-葡萄糖苷酶固定化酶。本发明的有益之处在于：该制备方法不仅工艺简单、成本低廉,而且制备得到的β-葡萄糖苷酶固定化酶在使用过程中稳定性好、催化效率高、可重复和连续利用。(The invention discloses a preparation method of beta-glucosidase immobilized enzyme, which comprises the following steps: placing the pretreated macroporous resin in a container, adding PBS (phosphate buffer solution) into the container, balancing, adding a beta-glucosidase zymogen solution, placing the container in a constant-temperature oscillation incubator for oscillation adsorption, separating the macroporous resin from the rest liquid after adsorption is finished, washing the separated macroporous resin with the PBS buffer solution until no protein can be detected in washing liquid, then adding the PBS buffer solution and a glutaraldehyde reagent for crosslinking, washing the macroporous resin with the PBS buffer solution for a plurality of times after crosslinking is finished, and drying under reduced pressure to obtain the beta-glucosidase immobilized enzyme. The invention has the advantages that: the preparation method has the advantages of simple process and low cost, and the prepared beta-glucosidase immobilized enzyme has good stability and high catalytic efficiency in the using process, and can be repeatedly and continuously utilized.)

1. A preparation method of a beta-glucosidase immobilized enzyme is characterized by comprising the following steps:

step 1: placing the pretreated macroporous resin in a container, adding a PBS buffer solution into the container, adding a beta-glucosidase zymogen solution after balancing, and then placing the container in a constant-temperature shaking incubator for shaking adsorption;

step 2: after adsorption, separating the macroporous resin from the rest liquid, washing the separated macroporous resin with PBS (phosphate buffer solution) until no protein can be detected in the washing liquid, and then adding PBS (phosphate buffer solution) and glutaraldehyde reagent for crosslinking;

step 3: after the crosslinking, the macroporous resin is washed by PBS buffer solution for a plurality of times, the beta-glucosidase immobilized enzyme is obtained after decompression and drying, and the beta-glucosidase immobilized enzyme is stored for standby at 4 ℃.

2. The method for preparing beta-glucosidase immobilized enzyme of claim 1, wherein in Step1, the macroporous resin is weak base anion exchange resin with primary amine group.

3. The method for preparing beta-glucosidase immobilized enzyme of claim 2, wherein the weak base anion exchange resin with primary amine group is D315 resin or D914 resin.

4. The method for preparing a beta-glucosidase immobilized enzyme of claim 3, wherein the amount of D315 resin or D914 resin added is 0.1-0.4g in 100mL of phosphate reaction system.

5. The method of producing a β -glucosidase immobilized enzyme of claim 1, wherein in Step1, the temperature of the incubator is normal temperature and the shaking time is 4 to 8 hours.

6. The method for preparing a beta-glucosidase immobilized enzyme as claimed in claim 1, wherein the amount ratio of the macroporous resin to glutaraldehyde in Step2 is 100-500 g: 1 mL.

7. The method for preparing a beta-glucosidase immobilized enzyme as claimed in claim 1, wherein the reaction temperature of crosslinking reaction is 30-40 ℃ and the reaction time is 0.5-2h in Step 2.

Technical Field

The invention relates to a preparation method of an immobilized enzyme, in particular to a preparation method of a beta-glucosidase immobilized enzyme, belonging to the technical field of biochemistry.

Background

Beta-glucosidase (EC 3.2.1.21), also known as beta-D-glucoside hydrolase, also known as cellobiase, gentinase and amygdalase, is known as beta-glucosidase in English and belongs to hydrolase.

Beta-glucosidase is a ubiquitous enzyme, is discovered in almond juice for the first time, is widely distributed in bacteria, fungi and plants, is an effective catalyst for hydrolyzing aromatic precursor substances and releasing conjugated aglycone, is an enzyme capable of catalyzing and hydrolyzing glycosyl or glycosyl bond between alkyl and glycosyl, is widely applied to various fields of medical treatment, food, biomass conversion and the like, and is also used as a high-stability and active catalyst for industrial food and biological processing.

Although the beta-glucosidase has the advantages of high catalytic efficiency, strong specificity, no environmental pollution and the like, the beta-glucosidase is expensive, poor in stability and difficult to recover, and the problems restrict the wide application of the beta-glucosidase in industry.

The enzyme is fixed on the solid carrier by using the enzyme immobilization technology, so that the enzyme can be recycled repeatedly, and the catalytic activity of the enzyme is retained because the catalytic group of the enzyme can be protected. Compared with free enzyme, the immobilized enzyme has the advantages of good stability, easy separation from a substrate and a product, easy control of catalytic reaction, repeated and continuous use and the like, can reduce the catalytic cost of the enzyme, and is widely applied to industrial production.

Disclosure of Invention

The invention aims to provide a preparation method of a beta-glucosidase immobilized enzyme, which has the advantages of simple process and low cost, and the prepared beta-glucosidase immobilized enzyme has good stability and high catalytic efficiency in the using process, and can be repeatedly and continuously utilized.

In order to achieve the above object, the present invention adopts the following technical solutions:

a preparation method of a beta-glucosidase immobilized enzyme is characterized by comprising the following steps:

step 1: placing the pretreated macroporous resin in a container, adding a PBS buffer solution into the container, adding a beta-glucosidase zymogen solution after balancing, and then placing the container in a constant-temperature shaking incubator for shaking adsorption;

step 2: after adsorption, separating the macroporous resin from the rest liquid, washing the separated macroporous resin with PBS (phosphate buffer solution) until no protein can be detected in the washing liquid, and then adding PBS (phosphate buffer solution) and glutaraldehyde reagent for crosslinking;

step 3: after the crosslinking, the macroporous resin is washed by PBS buffer solution for a plurality of times, the beta-glucosidase immobilized enzyme is obtained after decompression and drying, and the beta-glucosidase immobilized enzyme is stored for standby at 4 ℃.

The preparation method of the beta-glucosidase immobilized enzyme is characterized in that in Step1, the macroporous resin is weak-base anion exchange resin with primary amine groups.

The preparation method of the beta-glucosidase immobilized enzyme is characterized in that the weak base anion exchange resin with primary amine groups is D315 resin or D914 resin.

The preparation method of the beta-glucosidase immobilized enzyme is characterized in that the addition amount of the D315 resin or the D914 resin is 0.1-0.4g in a 100mL phosphate reaction system.

The method for producing a β -glucosidase immobilized enzyme is characterized in that in Step1, the temperature of the constant temperature shaking incubator is normal temperature, and the shaking time is 4 to 8 hours.

The preparation method of the beta-glucosidase immobilized enzyme is characterized in that in Step2, the dosage ratio of the macroporous resin to the glutaraldehyde is 100-500 g: 1 mL.

The preparation method of the beta-glucosidase immobilized enzyme is characterized in that in Step2, the crosslinking reaction temperature is 30-40 ℃, and the reaction time is 0.5-2 h.

The invention has the advantages that:

(1) the method of adsorption first and crosslinking second is utilized, namely, the beta-glucosidase is adsorbed on the surface of the macroporous resin through the electrostatic attraction effect first and then is crosslinked through glutaraldehyde to form the beta-glucosidase immobilized enzyme, and the prepared beta-glucosidase immobilized enzyme has good stability and high catalytic efficiency in the using process and can be repeatedly and continuously utilized, so that the industrial cost can be effectively reduced;

(2) the method of first adsorption and then crosslinking is utilized, the process is simple, and the cost is low.

Drawings

FIG. 1 is a temperature stability curve of a beta-glucosidase free enzyme and an immobilized enzyme;

FIG. 2 is a graph of the reusability of beta-glucosidase immobilized enzyme;

FIG. 3 is a graph showing the enzyme activity of the immobilized beta-glucosidase enzyme.

Detailed Description

The invention is described in detail below with reference to the figures and the embodiments.