Biological preparation method of gamma-aminobutyric acid

A technology for the preparation of aminobutyric acid and biology, applied in the field of biochemistry, can solve the problems of complex purification of γ-aminobutyric acid, high price of L-glutamic acid, and low price of sodium glutamate, and achieve complete conversion of raw materials, Environmentally friendly process and simple post-processing effect

Active Publication Date: 2019-05-10
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, glutamic acid decarboxylase needs to be active in an acidic environment, and the introduction of high concentration buffer will complicate the purification of GABA
Usually, high-purity L-glutamic acid is more expensive
Monosodium glutamate is less expensive, but the conversion rate is low

Method used

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  • Biological preparation method of gamma-aminobutyric acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1 prepares L-glutamic acid with monosodium glutamate

[0030] Dissolve 30g of monosodium glutamate in 80mL of water, add 14mL of concentrated hydrochloric acid to convert the monosodium glutamate into L-glutamic acid and precipitate it from the water, wash the filtered L-glutamic acid with water for several times until the pH of the washed liquid is greater than 3 Stop washing when the time comes, collect L-glutamic acid, and use it in the reaction after drying.

Embodiment 2

[0031] Example 2 Preparation of glutamic acid decarboxylase whole cell

[0032] Inoculate the Gad-pET28a recombinant Escherichia coli engineering strain into SOB liquid medium, which contains 50mg / L of kanamycin; after inoculation, shake the flask on a shaker at 37°C and 200rpm, when the OD of the bacteria 600 When it reached 0.6, it was transferred to a 5L fermenter containing 2L SOB medium, and the inoculation ratio was 10%. The fermentation temperature in the early stage of the reaction is 37°C, the enzyme production temperature in the later stage is 25°C, the stirring speed is 400rpm, the aeration ratio and tank pressure are 2VVM (cubic meter / (cubic meter*min)) and 0.05MPa respectively; Sampling was taken to monitor the cell mass and determine the enzyme activity. When the cell mass and enzyme activity remained stable, the fermentation was stopped, and the resting cells were collected by centrifugation at 4°C (6000rpm, 5min), and the wet cell mass reached 60g / L.

Embodiment 3

[0033] Embodiment 3 prepares gamma-aminobutyric acid

[0034] 500mL reaction system to prepare the compound γ-aminobutyric acid: add 500mL water to a 1L reaction kettle, add 13g whole cells of glutamic acid decarboxylase, add pyridoxal phosphate with a final concentration of 0.5mM, and add L-glutamic acid at a time 100g, react at 35-40°C, 220rpm for 7 hours, use thin-layer chromatography to detect the progress of the end point of the reaction, add 100g of L-glutamic acid when the substrate is reacted, repeat in sequence, and add L-glutamic acid in total After 350 g and complete reaction, the supernatant was collected by centrifugation.

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Abstract

The invention provides a biological preparation method of gamma-aminobutyric acid, which comprises the following steps of: 1) preparing glutamic acid decarboxylase whole cell thallus by using an E. coli engineering strain capable of expressing glutamic acid decarboxylase; 2) using monosodium glutamate and concentrated hydrochloric acid to prepare L-glutamic acid; 3) dissolving L-glutamic acid as asubstrate in water, adding glutamic acid decarboxylase whole cell thallus and pyridoxal phosphate, stirring and reacting to obtain gamma-aminobutyric acid. The method uses water to replace buffer salt solution to form a water-phase reaction system, uses monosodium glutamate to replace high-purity glutamic acid, so that the purity of the gamma-aminobutyric acid is up to 97.7 percent, and the costof raw materials is reduced; whole cells used for catalysis can be produced in a large scale by microbial fermentation, the cost is low, and the source is wide; the biological enzyme-promoted catalytic reaction is a water-phase reaction, the enzymatic conversion condition is mild, the raw material conversion is thorough, the post-treatment is simple, the product is separated by adopting a method of evaporation concentration and natural crystallization, the cost is low, the process is environment-friendly, and the method is suitable for industrial production of gamma-aminobutyric acid.

Description

technical field [0001] The invention belongs to the field of biochemistry, and in particular relates to a biological preparation method of gamma-aminobutyric acid. Background technique [0002] γ-aminobutyric acid is an important neuroinhibitory mediator in the central nervous system of mammals, mediating more than 40% of neuroinhibitory signals. γ-aminobutyric acid plays extremely important physiological functions in the human body, such as promoting brain activation, calming, anticonvulsant, antiepileptic, promoting sleep, delaying brain aging, replenishing human inhibitory neurotransmitters, lowering blood pressure, and at the same time Can inhibit fatty liver and obesity, activate liver function, etc. In addition, γ-aminobutyric acid can be used as an environmentally friendly precursor for the industrial synthesis of nitrogen-containing chemicals such as N-methylpyrrolidone, bioplastics, and nylon. Therefore, it plays a very important role in food, pharmaceutical, chem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/60C12P13/00
Inventor 马立新王亚平刘洋
Owner HUBEI UNIV
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