Method for preparing glutamate decarboxylase (GAD)

A technology of glutamic acid decarboxylase and solution is applied in the field of bioengineering and achieves the effects of simple preparation process, low production cost and high recovery rate of enzyme activity

Inactive Publication Date: 2013-09-18
QILU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The GAD molecules produced by microorganisms are relatively large. When reverse micelles are used for extraction and separation, a new reverse micelles system must be designed so that the formed reverse micelles have a larger internal space size to meet the requirements for dissolving GAD molecules. Therefore, there is no report on the preparation of glutamic acid decarboxylase by reverse micelles extraction

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] A method for preparing glutamic acid decarboxylase by using a reverse micelle extraction system, the steps are as follows:

[0055] (1) Centrifuge 1000 mL of Escherichia coli somatic cell culture solution containing glutamic acid decarboxylase at 3000 r / min for 10 minutes to obtain wet bacterial cells, resuspend the wet bacterial cells in deionized water, and prepare Obtain a suspension with a cell mass concentration of 25 wt%, cool it to about 10°C, and use the APV-2000-1 high-pressure cell crusher produced by the German APV company to perform three cell disruptions. The operating pressure of the crusher is 60MPa to obtain a cell disruption solution. 312mL. The pH value of the cell disruption solution was adjusted to 5.5 with hydrochloric acid, and then potassium chloride was added to adjust the ionic strength of the solution to 0.10 mol / L to obtain a cell disruption solution containing glutamic acid decarboxylase.

[0056] (2) Add 100mL n-butanol to 400mL isooctane, ...

Embodiment 2

[0063] Utilize the method for preparing glutamic acid decarboxylase described in embodiment 1 by reverse micelle extraction system, difference is:

[0064] In step (1), adjust the pH value of the cell disruption solution to 7.0; then add potassium chloride to adjust the ionic strength of the solution to 0.20 mol / L to prepare a cell disruption solution containing glutamic acid decarboxylase.

[0065] In step (2), CTAB and Span80 were added to the n-butanol-isooctane solution so that the concentration of the surfactant in the organic solution was 150 mmol / L.

[0066]In step (2), prepare 400mL of 0.10mol / L citric acid-sodium citrate buffer solution (pH4.2), and then add isopropanol to the buffer solution so that the mass concentration in the buffer solution is 25wt% , shake well, and then add potassium chloride to adjust the ionic strength of the solution to 1.0 mol / L to prepare an aqueous stripping solution.

[0067] The enzyme activity recovery rate of glutamic acid decarboxyl...

Embodiment 3

[0069] Utilize the method for preparing glutamic acid decarboxylase described in embodiment 1 by reverse micelle extraction system, difference is:

[0070] In step (3), the cell disruption solution is added to the CTAB-Span80 / n-butanol-isooctane reverse micelles system solution, and the volume ratio of the mixture is: the volume of the reverse micelles system solution: the volume of the cell disruption solution =1:3.

[0071] In step (4), the stripping aqueous phase solution is added to the reverse micelles solution containing glutamic acid decarboxylase, and the volume ratio of the mixed solution is: the volume of the stripping aqueous phase solution: the reverse gum containing glutamic acid decarboxylase The volume of the mass solution = 1:10.

[0072] The enzyme activity recovery rate of glutamic acid decarboxylase was detected to reach 91.2%, and the purification factor reached about 3.1.

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Abstract

The invention relates to a method for preparing glutamate decarboxylase (GAD). The method comprises the following steps: (1) culturing a microbial cell containing GAD, centrifugally collecting the cell, disrupting the cell, and regulating the pH value and ionic strength of cell disruption solution; (2) preparing a reverse micelle extraction system solution and a reverse extraction aqueous phase solution; (3) utilizing reverse micelle for extraction; (4) carrying out reverse extraction to obtain an aqueous solution containing GAD; and (5) carrying out filtration and concentration with an ultrafiltration membrane, and freeze-drying trapped fluid, thus preparing the GAD product. The method has the beneficial effects that continuous and large-scale preparation of GAD can be achieved; the preparation process of GAD is simple and convenient, and the production cycle is short; the activity and recovery rate of GAD are high; the extraction agent can be recycled; and the production cost of GAD is lower.

Description

technical field [0001] The invention relates to a method for preparing glutamic acid decarboxylase, in particular to a method for preparing glutamic acid decarboxylase by reverse micelles extraction technology, and belongs to the technical field of bioengineering. Background technique [0002] Glutamate decarboxylase (Glutamate decarboxylase, GAD) is a kind of pyridoxal lyase, which widely exists in plants, animals and microorganisms. Glutamate decarboxylase biocatalyzes the decarboxylation of the α-carboxyl group on L-glutamic acid or its sodium salt to generate γ-aminobutyric acid and CO 2 It is also very likely to be used as a specific diagnostic enzyme to predict and distinguish diabetes and as a very potential diagnostic and therapeutic enzyme preparation. [0003] The γ-aminobutyric acid catalyzed by glutamic acid decarboxylase has many physiological functions such as lowering blood pressure, enhancing brain vitality, nourishing nerve cells, maintaining nerve stabilit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12R1/19C12R1/01
Inventor 董永胜
Owner QILU UNIV OF TECH
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