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Immobilized glutamate decarboxylase and preparation method thereof

A technology of glutamic acid decarboxylase and acetic acid, which is applied in the direction of immobilization on/in the organic carrier, can solve the problem of weak combination of glutamic acid decarboxylase and carrier, complicated immobilization materials and methods, and unsatisfactory results, etc. problem, to achieve the effect of simple immobilization method, long catalytic reaction life and large loading capacity

Inactive Publication Date: 2011-07-13
山东水晶生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, for the immobilization method of glutamic acid decarboxylase and the research of immobilized glutamic acid decarboxylase, there are a small number of literature reports, but or immobilization materials and methods are complicated, or glutamic acid decarboxylase is not firmly combined with the carrier, or Using commercially available glutamic acid decarboxylase acetone powder as the enzyme source, the activity after immobilization is low, and the results are not ideal

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1, preparation immobilized glutamic acid decarboxylase

[0030] 1) Prepare glutamic acid decarboxylase crude enzyme solution. Add 20mL / g to the wet cells of lactic acid bacteria NBRC 12005 湿菌体 acetic acid-acetate buffer (0.2mol / L, pH 4.6), suspend the bacteria in the centrifuge tube (the bacteria suspension contains 350 cfu / ml), and ultrasonically break it under the condition of ice-water bath, and the ultrasonic treatment condition is ultrasonic The power is 300W, the working time is 6s, the intermittent time is 9s, and the number of ultrasonic work is 30 times. After the sonication, the crude enzyme solution of glutamic acid decarboxylase is obtained.

[0031] 2) Pretreatment cotton flannelette carrier:

[0032] a) Use 10 mL of 0.5 mol / L sodium hydroxide solution at 30°C to treat a 3cm×3cm lint cloth for 3 hours, blot the treated lint cloth with absorbent paper, and then dry it at 50°C.

[0033] b) Use 10mL / g 干棉绒布 Treat the cotton flannelette obtained ...

Embodiment 2

[0040] Embodiment 2, preparation immobilized glutamic acid decarboxylase

[0041] 1) Prepare glutamic acid decarboxylase crude enzyme solution. Add 20mL / g to the wet cells of lactic acid bacteria NBRC 12005 湿菌体 acetic acid-acetate buffer solution (0.4mol / L, pH 5.0), suspend the bacteria in the centrifuge tube (the bacterial suspension contains 400cfu / ml), and ultrasonically break it under the condition of ice-water bath, and the ultrasonic treatment condition is ultrasonic The power is 200W, the working time is 8s, the intermittent time is 7s, and the number of ultrasonic work is 30 times. After the sonication, the crude enzyme solution of glutamic acid decarboxylase is obtained.

[0042] 2) Pretreatment cotton flannelette carrier:

[0043] a) Use 10 mL of 0.8 mol / L sodium hydroxide solution to treat a 3 cm x 3 cm cotton flannelette at 40°C for 4 hours, blot the treated cotton flannelette dry with absorbent paper, and then dry it at 60°C.

[0044] b) Use 5mL / g 干棉绒布 Dry pyri...

Embodiment 3

[0050] Embodiment 3, preparation immobilized glutamic acid decarboxylase

[0051] 1) Prepare glutamic acid decarboxylase crude enzyme solution. Add 20mL / g to the wet cell of Lactobacillus brevis 湿菌体 acetic acid-acetate buffer solution (0.1mol / L, pH 4.0), suspend the bacteria in the centrifuge tube (the bacteria suspension contains 500 cfu / ml), and ultrasonically break it under the condition of ice-water bath, and the ultrasonic treatment condition is ultrasonic The power is 400W, the working time is 6s, the intermittent time is 11s, and the number of ultrasonic work is 30 times. After the sonication, the crude enzyme solution of glutamic acid decarboxylase is obtained.

[0052] 2) Pretreatment cotton flannelette carrier:

[0053] a) Use 10 mL of 0.2 mol / L sodium hydroxide solution to treat 3 cm x 3 cm cotton flannelette at 5°C for 5 hours, blot the treated cotton flannelette dry with absorbent paper, and then dry it at 60°C.

[0054] b) Use 20mL / g 干棉绒布 Dry pyridine to tre...

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Abstract

The invention discloses an immobilized glutamate decarboxylase and a preparation method thereof. The immobilized glutamate decarboxylase is prepared according to the method comprising the following steps of: 1) adding acetic acid-acetate buffer solution into lactic acid bacteria to obtain bacterial suspension containing 300-500cfu bacteria per milliliter, performing ultrasonic crushing on the bacterial suspension under the condition of an ice-water bath to obtain crude enzyme solution of the glutamate decarboxylase; 2) using cotton flannel fabric as a carrier, and sequentially performing pretreatment with sodium hydroxide, pyridine and para-toluenesulfonyl chloride; and 3) arranging the cotton flannel fabric after the pretreatment in the crude enzyme solution of the glutamate decarboxylase for soaking, adding glutaraldehyde into the solution, and realizing immobilization of the glutamate decarboxylase. The immobilization method of the glutamate decarboxylase is simple, the conditions are required to be mild and easy to implement, and the obtained immobilized glutamate decarboxylase has the advantages of high activity, large loading amount, long catalytic reaction service life and the like.

Description

technical field [0001] The invention relates to an immobilized glutamic acid decarboxylase and a preparation method thereof. Background technique [0002] Gamma-aminobutyric acid, a non-protein-forming amino acid, is an important inhibitory neurotransmitter in the nervous system of mammals, crustaceans, insects and some parasitic worms, and some plants also contain this amino acid. In the human brain, GABA is produced by the conversion of glutamate by glutamate decarboxylase in the brain. γ-aminobutyric acid in organisms can participate in physiological activities such as analgesia, anti-anxiety, anti-convulsion, and lowering blood pressure, and has various biological functions. [0003] The preparation of γ-aminobutyric acid is generally divided into three categories: chemical synthesis, plant enrichment extraction and biosynthesis. Among them, there are many methods for chemically synthesizing γ-aminobutyric acid, but the cost is high, the reaction conditions are not eas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/04
Inventor 孙君社刘萍张秀清范杰张京声
Owner 山东水晶生物科技股份有限公司
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