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Enzyme engineering method for producing gamma-aminobutyric acid

A technology of aminobutyric acid and enzyme engineering, which is applied in the field of enzyme production by fermentation, can solve the problems of high risk, risk in the operation process, high production cost, etc., and achieve the effect of reducing the discharge of three wastes

Inactive Publication Date: 2011-11-16
山东恩贝生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Both pyrrolidone and sodium hydroxide are corrosive substances that affect human health
In particular, pyrrolidone has the danger of burning at high temperatures, and can decompose and release toxic nitrogen oxide fumes. The operation process is dangerous, which is not conducive to large-scale industrial production.
The solid-state fermentation method is inefficient, the output is only 0.35mg / g, the final purity is only 45%, the production cost is high, and there is no market competitiveness
The plant extraction method uses organic solvents to extract from plants. Organic solvents are dangerous, flammable and explosive, and the content of γ-aminobutyric acid in plants is low, while the content of impurities is high, resulting in the production of γ-aminobutyric acid by plant extraction methods. Low acid yield and low product purity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Introduce strains into the culture medium for routine culture. The composition of the culture medium is that the components contained in each liter of culture solution are in grams: corn steep liquor 10.0, yeast powder 15.0, yeast extract 10.0, KH 2 PO 4 2.0, MgSO 4 0.5, the inducer lactose is sterilized separately at a concentration of 50% (making the final concentration added to the fermenter be 0.01%). Add the prepared medium into the fermenter and sterilize at 121°C for 30 minutes. The strains were inoculated by flame inoculation method, and the inoculum amount was 3%. The fermentation time is 18 hours, and the inducer is gradually added at 12 hours to prepare the bacterial liquid;

[0016] Small test enzyme activity detection: Take 100ml of bacterial liquid for centrifugation, centrifuge the bacterial cells and discard the supernatant, add 0.1mol / L acetic acid-sodium acetate buffer solution with a pH of 5.0, mix the bacterial cells, add Tween 800.1 ml, and th...

Embodiment 2

[0019] Introduce Escherichia coli into the culture medium for routine culture in tanks. The composition of the culture medium is that the components contained in each liter of culture solution are in g: corn steep liquor 10.0, yeast powder 15.0, yeast extract 10.0, KH 2 PO 4 2.0, MgSO 4 0.5, adding 0.2% lactose of medium mass as an inducer to induce enzyme production, the inoculum size is 3%, the culture temperature is 35-39°C, the pH is 5.0, cultured for 24 hours, and the bacterial liquid is obtained, and the volume of the tank is 15t.

[0020] Small test enzyme activity detection: take 100ml of bacterial liquid for centrifugation, centrifuge out the bacterial cells and discard the supernatant, add 0.1mol / L sodium acetate-acetic acid buffer solution with a pH of 5.0, mix the bacterial cells, add Tween 80 0.03ml, add 10g of glutamic acid for conversion. The reaction temperature is 40°C, and the reaction time is 36h. The residual glutamic acid was detected to be 0.2 g / L, a...

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Abstract

The invention discloses an enzyme engineering method for producing gamma-aminobutyric acid, belonging to the technical field of a fermentation method for producing enzymes. The method disclosed by the invention comprises the following steps of: inducing by using lactose as an inducer for producing enzymes, filtering through a ceramic film for collecting thalli, and washing the thalli instantly; then, adding an acetic acid-sodium acetate buffer solution into the thalli, and adding a small amount of substrate glutamic acid many times to enable the glutamic acid to be converted into gamma-aminobutyric acid; then, filtering and removing the thalli instantly by adopting the ceramic film, and decoloring by adopting a rolled organic film; and finally, carrying out triple-effect concentration, stopping concentrating until the content of the concentrated product reaches 550-600g / L, pouring the concentrated solution into a vacuum concentration crystallizing tank, continuing to concentrate, stopping heating until the content of the product reaches 700-900g / L, and naturally cooling and growing the grain for 1-1.5 hours under the normal pressure to obtain a columnar or powdered crystal. The purity of the crystal obtained by primary crystallization is greater than or equal to 99.0%, and the yield of the crystal is greater than or equal to 85%. The method disclosed by the invention is a clean production process and has low emission of the three wastes, and the residues of the thalli can be used as feed proteins.

Description

technical field [0001] The invention relates to a method for producing gamma-aminobutyric acid by using an enzyme engineering method, and belongs to the technical field of enzyme production by fermentation. Background technique [0002] γ-aminobutyric acid (γ-aminobutyric acid, GABA for short), also known as aminobutyric acid, chemical name 4-aminobutyric acid, gamma aminobutyric acid. Appearance is almost white crystal or crystalline powder, slight odor, strong hygroscopicity. GABA has the functions of stabilizing, anticonvulsant, lowering blood pressure, enhancing brain vitality, nourishing nerve cells, promoting secretion of growth hormone, protecting liver and kidney. The results of research on the application of GABA in livestock and poultry breeding industry show that GABA has good anti-heat stress and growth-promoting effects on livestock and poultry. [0003] The production methods of γ-aminobutyric acid mainly include chemical synthesis, solid-state fermentation a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/00C12R1/19
Inventor 郑雄敏王震荣金雷
Owner 山东恩贝生物工程有限公司
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