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Preparation method of beta-glucosaccharase cross-linked aggregates

A technology of glucosidase and cross-linking polymerization, which is applied in the field of preparation of β-glucosidase cross-linked aggregates, can solve problems such as low recovery rate of enzyme activity, and achieve the effect of improving recovery rate of enzyme activity

Inactive Publication Date: 2014-03-26
FUZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The present invention aims to provide a preparation method of β-glucosidase cross-linked aggregates (β-glucosidase CLEAs), the β-glucosidase CLEAs prepared by this method can solve the problem of immobilized glycosides in the prior art The problem of low recovery rate of enzyme activity in industrial production process

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] A preparation method of β-glucosidase cross-linked aggregates (β-glucosidase CLEAs), comprising the following steps: (1) adding 2.5ml 1,4-dioxane to 1 mL of β-glucosidase containing Enzyme 1.5mg / ml β-glucosidase solution (that is, the volume ratio of 1,4-dioxane to β-glucosidase solution is 2.5:1), shake; (2) Add 25% glutaraldehyde , until the final concentration of glutaraldehyde is 43mM, and the crosslinking time is 10min; (3) add sodium borohydride (NaBH 4 ), to NaBH 4 The final concentration of β-glucosidase CLEAs was reduced to 25 mM, shaken, and centrifuged at 400 rpm for 5 minutes to collect the precipitates, which were β-glucosidase CLEAs; the reaction temperature was controlled at 5°C during the entire reaction process, and the pH value was 5.5.

[0015] The recovery and processing method of β-glucosidase CLEAs is as follows: after centrifugation of the reaction solution, the collected β-glucosidase CLEAs precipitate is washed with buffer solution for 3 times,...

Embodiment 2

[0020] The difference between Example 2 and Example 1 is that the pH value during the entire reaction process is 5.0, wherein the volume of 1,4-dioxane in step (1) is 2ml (that is, 1,4-dioxane and The volume ratio of β-glucosidase solution is 2:1), the β-glucosidase solution contains β-glucosidase 2.5mg / ml; the final concentration of glutaraldehyde in step (2) is 40 mM; step (3 ) in NaBH 4 The final concentration of β-glucosidase CLEAs was 23 mM, centrifuged at 300rpm for 10min; the recovery rate of the enzyme activity of β-glucosidase CLEAs was 81.78%.

Embodiment 3

[0022] The difference between Example 3 and Example 1 is that the pH value during the entire reaction process is 5.2, wherein the volume of 1,4-dioxane in step (1) is 3ml (that is, 1,4-dioxane and The volume ratio of β-glucosidase solution is 3:1), β-glucosidase solution contains β-glucosidase 2.0mg / ml; the final concentration of glutaraldehyde in step (2) is 38 mM; step (3 ) in NaBH 4 The final concentration of β-glucosidase CLEAs was 20mM, centrifuged at 500rpm for 5min, and the recovery rate of enzyme activity of β-glucosidase CLEAs was 80.11%.

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PUM

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Abstract

The invention provides a preparation method of beta-glucosaccharase cross-linked aggregates, which can obtain a high-enzyme-activity recovery rate. The method comprises the following steps: (1) adding a settling agent into a beta-glucosaccharase solution and oscillating; (2) adding glutaraldehyde; (3) adding a reducing agent into a mixed solution, oscillating, centrifuging and collecting precipitate, wherein the reaction temperature of the entire reaction process is controlled at 4-6 DEG C, and the pH value is 5.0-5.5; the settling agent used in the step (1) is 1,4-dioxane, and the volume ratio of 1,4-dioxane to the beta-glucosaccharase solution is (2-3):1; the concentration of glutaraldehyde in the step (2) is 38-43mM, and crosslinking time is 8-12 minutes; the reducing agent is sodium borohydride (NaBH4), and the concentration of NaBH4 is 20-25mM.

Description

technical field [0001] The invention belongs to the field of immobilized enzyme production, and in particular relates to a preparation method of a β-glucosidase cross-linked aggregate. Background technique [0002] Free β-glucosidase has problems such as poor stability and low reuse rate in industrial production, which seriously restricts the industrial production of biosynthetic salidroside. The cross-linked enzyme aggregate does not require crystallization of the enzyme, but also can maximize the recovery of the enzyme activity, which is the most excellent carrier-free immobilized enzyme technology. With the rapid development of cross-linked aggregate technology in the field of enzyme immobilization, in recent years, some people have studied the preparation method of β-glucosidase cross-linked aggregates (see: Ou Jie, cross-linked β-glucosidase aggregates Preparation and production of active aglycones of soybean isoflavones, Hefei University of Technology, master thesis, ...

Claims

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Application Information

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IPC IPC(8): C12N9/42
CPCC12N9/2445C12Y302/01021
Inventor 石贤爱薛原楷
Owner FUZHOU UNIVERSITY
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