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Method for immobilizing escherichia coli glutamate decarboxylase through entrapping method

A technology of glutamate decarboxylase and Escherichia coli, applied in the field of bioengineering, can solve the problems of weak binding of glutamate decarboxylase and carrier, unsatisfactory effect, low enzyme activity, etc., and achieves controllable size and uniform particle size. , the effect of improving enzyme activity

Inactive Publication Date: 2015-04-22
SOOCHOW KH BIO SCI & TECH CO LTD KHB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, glutamic acid deacidase is not firmly combined with the carrier. According to existing literature reports, the enzyme activity recovery rate of the sodium alginate immobilization method is as high as 65.%, and the enzyme activity after immobilization is low, and the effect is not ideal.

Method used

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  • Method for immobilizing escherichia coli glutamate decarboxylase through entrapping method
  • Method for immobilizing escherichia coli glutamate decarboxylase through entrapping method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Sodium alginate solution composition: sodium alginate 4%, glycerin 15%, polyethylene glycol octylphenyl ether (100) 1.5%, polyethylene glycol (8000) 0.75%, dithiothreitol 0.5mM, CaCl 2 The solution concentration is 5%.

[0022] The specific activity of Escherichia coli glutamic acid decarboxylase enzyme solution is 150U / mg, and the protein concentration is 0.5mg / mL.

[0023] Preparation method of immobilized glutamic acid decarboxylase:

[0024] (1) Sodium alginate carrier preparation: Soak sodium alginate with glycerin, then add deionized water, stir to dissolve, add polyethylene glycol octylphenyl ether, polyethylene glycol, dithiothreitol, stir Dissolve to obtain a uniform sodium alginate solution; (2) add the glutamic acid decarboxylase enzyme solution to the sodium alginate solution prepared in step (1), and mix the two evenly to obtain a sodium alginate enzyme solution; (3) use a constant The flow pump is externally connected to the syringe needle, and the sodiu...

Embodiment 2

[0028] Sodium alginate solution composition: sodium alginate 4%, glycerin 20%, polyethylene glycol octylphenyl ether 1.5%, polyethylene glycol 1%, dithiothreitol 1mM.

[0029] CaCl 2 The solution concentration is 6%.

[0030] The specific activity of Escherichia coli glutamic acid decarboxylase enzyme solution is 150U / mg, and the protein concentration is 0.5mg / mL.

[0031] The preparation method of immobilized glutamic acid decarboxylase is the same as in Example 1.

[0032] The recovery rate of glutamic acid decarboxylase was measured to be 73.5%.

Embodiment 3

[0034] The composition of the sodium alginate solution: 3% sodium alginate, 10% glycerin, 1.5% polyethylene glycol octylphenyl ether, 1% polyethylene glycol, and 1 mM dithiothreitol.

[0035] CaCl 2 Solution concentration 2%.

[0036] The specific activity of Escherichia coli glutamic acid decarboxylase enzyme solution is 150U / mg, and the protein concentration is 0.5mg / mL.

[0037] The preparation method of immobilized glutamic acid decarboxylase is the same as in Example 1.

[0038] The recovery rate of glutamic acid decarboxylase was measured to be 65%.

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Abstract

The invention discloses a method for immobilizing escherichia coli glutamate decarboxylase through an entrapping method, wherein the method comprises the following steps: (1) preparing a sodium alginate carrier: infiltrating sodium alginate with glycerol, then adding deionized water, stirring to dissolve, then adding polyoxyethylene octyl phenyl ether, polyethylene glycol and dithiothreitol, stirring to dissolve, and thus obtaining a homogeneous sodium alginate solution; (2) adding a glutamate decarboxylase enzyme solution into the sodium alginate solution prepared in the step (1), mixing the two solutions uniformly, and thus obtaining a sodium alginate enzyme solution; (3) dropwise adding the sodium alginate enzyme solution into a CaCl2 solution with a needle of a constant-flow pump externally-connected injector at a uniform velocity, stirring and immobilizing; and (4) filtering, repeatedly washing a gel precipitate with a PBS buffer solution, drying, and thus obtaining granular immobilized glutamate decarboxylase. Glycerol, polyoxyethylene octyl phenyl ether, polyethylene glycol and dithiothreitol are added in the sodium alginate solution, and the enzyme activity of the glutamate decarboxylase after immobilization is effectively improved.

Description

technical field [0001] The invention relates to the immobilization of enzymes, in particular to a method for immobilizing Escherichia coli glutamic acid decarboxylase by an embedding method, and belongs to the field of bioengineering. Background technique [0002] Gamma-aminobutyric acid (GABA) exists in the brain and spinal cord of mammals, and is an inhibitory neurotransmitter, which is obtained from glutamic acid catalyzed by glutamic acid decarboxylase (glutamic acid decarboxylase). GABA has the functions of regulating blood pressure and heart rhythm, nourishing nerve cells, promoting mental stability, promoting blood supply to the brain, promoting long-term memory, promoting hormone secretion, and activating kidney function. [0003] The production of γ-aminobutyric acid basically adopts chemical synthesis, plant enrichment method and biosynthesis method. Among them, the cost of chemical synthesis is high and the safety is poor due to the use of toxic reagents, and the...

Claims

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Application Information

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IPC IPC(8): C12N11/10
Inventor 金京勋
Owner SOOCHOW KH BIO SCI & TECH CO LTD KHB
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