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Separating and purifying emthod for endo-beta-1,4-glucanase

A purification method and glucanase technology are applied in the field of separation and purification of endo-β-1 by electroprecipitation technology, which can solve the problems of serious loss of enzyme activity, unfavorable single enzyme component research and application of single enzyme component, etc. The effect of less loss, precise control of operating parameters and strong precipitation ability

Inactive Publication Date: 2006-04-26
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This purification process has disadvantages such as serious loss of enzyme activity, and the product is generally a mixture of various enzymes, which is not conducive to the research on the properties of a single enzyme component and the application of a single enzyme component

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] First prepare the ethanol-water solution of cellulase with a concentration of 1% (mass percentage), wherein the ethanol concentration is 25% (volume percentage), fully oscillate to make the solution mix evenly, then get 20mL of the solution and add it to a preheated to 25 ° C in an autoclave. Then, pressurized CO with a preheating temperature of 25°C was introduced. 2 , to increase the pressure to 4.5MPa, adjust the operating temperature to 25°C, and the pH of the solution is about 4.0 at this time. The pressure was maintained for 40 minutes in this state, and the endo-β-1,4-glucanase was completely precipitated under this condition. Endo-β-1,4-glucanase precipitation was obtained by filtration, and its purity and enzyme activity were measured. At this time, the purification multiple of endo-β-1,4-glucanase was 1.65, and the enzyme activity yield was 84.1%. This method uses pressurized CO 2 As a volatile acid, ethanol acts as a precipitant, forming pressurized CO 2...

Embodiment 2

[0015] First prepare the ethanol-water solution of cellulase with a concentration of 3% (mass percentage), wherein the ethanol concentration is 40% (volume percentage), fully oscillate to make the solution mix uniformly, then get 20mL of the solution and add it to a preheated to 20°C in an autoclave. Then, pressurized CO with a preheating temperature of 20°C was introduced. 2 , increase the pressure to 4.5MPa, adjust the operating temperature to 20°C, and keep the pressure in this state for 55 minutes, and the endo-β-1,4-glucanase in it will be completely precipitated under this condition. Endo-beta-1,4-glucanase was filtered to precipitate, and its purity and enzyme activity were measured. At this time, the purification multiple of endo-beta-1,4-glucanase was 1.43, and the enzyme activity yield was 89.2%. This method uses pressurized CO 2 As a volatile acid, ethanol acts as a precipitant, forming pressurized CO 2 -Ethanol-water system protein isoelectric precipitation tec...

Embodiment 3

[0017] First prepare the ethanol-water solution of cellulase with a concentration of 1% (mass percentage), wherein the ethanol concentration is 20% (volume percentage), fully oscillate to make the solution mix uniformly, then take 20mL of the solution and add it to a preheated to 20°C in an autoclave. Then, pressurized CO with a preheating temperature of 20°C was introduced. 2 , increase the pressure to 3.5MPa, adjust the operating temperature to 20°C, and keep the pressure for 30 minutes under this state, and the endo-β-1,4-glucanase in it will be completely precipitated under this condition. Endo-β-1,4-glucanase precipitate was obtained by filtration, and its purity and enzyme activity were measured. At this time, the purification multiple of endo-β-1,4-glucanase was 1.52, and the enzyme activity yield was 73.6%. This method uses pressurized CO 2 As a volatile acid, ethanol acts as a precipitant, forming pressurized CO 2 -Ethanol-water system protein isoelectric precipit...

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PUM

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Abstract

The invention discloses a method for separating and purifying endo-beta-1,4-glucanase. The steps of the method are as follows: 1) First, dissolve the crude cellulase in the ethanol-water solution, prepare a solution with a concentration of 1 to 3% by mass of the crude cellulase and 10 to 40% by volume of ethanol, and fully oscillate to make Mix the solution evenly; 2) Then, take 20mL of the above mixed solution and add it to an autoclave with a preheating temperature of 20-40°C, and then pass it into a pressurized CO with a preheating temperature of 20-40°C. 2 , increase the pressure to 35-8.0 MPa, adjust the pH to 3.8-4.2, keep it for 25-60 minutes, and filter to obtain endo-β-1,4-glucanase. The invention has the characteristics that the product obtained after separation can maintain high enzyme activity, and the water-soluble protein is easy to form precipitation. -The separation and purification method of glucanase.

Description

technical field [0001] The present invention relates to a separation and purification method, in particular to a method using pressurized CO 2 A method for separating and purifying endo-β-1,4-glucanase using isoelectric precipitation technology as a volatile acid and ethanol as a precipitating aid. Background technique [0002] Endo-β-1,4-glucanase is a kind of cellulase. Cellulase refers to the general term for a class of enzymes that degrade cellulose, and its greatest use is to enzymatically hydrolyze cellulose into glucose. Cellulose is the most abundant carbohydrate in nature, exceeding the sum of other carbohydrates, and is an important renewable resource and energy source. Therefore, the study of cellulase is considered to be the key to the development and utilization of renewable resources in the new century, and it is of great significance to solve the problems of industrial and agricultural raw material sources, energy crisis, and environmental pollution. Now th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42
Inventor 姚善泾关怡新齐祥明
Owner ZHEJIANG UNIV