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A kind of synthetic method of L-theanine

A synthetic method and theanine technology, applied in the field of bioengineering, can solve problems such as low efficiency, complex reaction system, unfavorable industrial production, etc.

Active Publication Date: 2020-11-17
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this ATP regeneration method uses the glycolysis pathway in yeast, which requires multiple steps, so the efficiency is relatively low; at the same time, the problem of cell membrane permeability needs to be considered in the reaction system
In addition, there are many factors affecting the metabolism of living cells in the reaction, which makes the reaction system complicated and is not conducive to industrial production.

Method used

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  • A kind of synthetic method of L-theanine
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  • A kind of synthetic method of L-theanine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Enzymatic synthesis of L-theanine

[0042] In a 50mL reaction flask, add the final concentrations of 100mM sodium glutamate, 100mM ethylamine hydrochloride, 150mM magnesium chloride hexahydrate, 75mM sodium hexametaphosphate, 5mM adenosine triphosphate (ATP) and 75mM imidazole buffer (pH 8.0) in sequence. , dissolve in water to make the final volume 10mL, adjust the reaction pH to 7.0 with 5M sodium hydroxide solution, add 50mg γ-glutamyl methylamine synthase and 40mg phosphokinase. After reacting at 37° C. and 200 rpm for 24 h, samples were taken for HPLC analysis.

[0043] The detection method is: TAG; UV 210nm detection; mobile phase: ammonium acetate buffer (5mM, pH 6.0) / methanol=25 / 75, v:v.

[0044] The results showed that the conversion rate of L-theanine was 93%, and the ee value was >99%.

Embodiment 2

[0045] Example 2 Enzymatic synthesis of L-theanine

[0046] Add the final concentration of 200mM sodium glutamate, 200mM ethylamine hydrochloride, 150mM magnesium chloride hexahydrate, 75mM sodium hexametaphosphate, 5mM disodium adenosine triphosphate (ATP) to the 100mL reaction flask in sequence, add water to dissolve to make the final volume 50mL, The pH of the reaction was adjusted to 7.0 with 5M sodium hydroxide solution, and 250 mg of gamma-glutamyl methylamine synthase and 200 mg of phosphokinase were added. The reaction was carried out at 37° C. under mechanical stirring, and the pH of the reaction was maintained at 7.0 with 2M sodium hydroxide solution during the reaction. After 7 hours of reaction, sampling was carried out for HPLC analysis. The detection method was the same as that in Example 1.

[0047] The results showed that the conversion rate was 90% and the ee value was >99%.

Embodiment 3

[0048] Example 3 Enzymatic synthesis of L-theanine

[0049] Add the final concentration of 400mM sodium glutamate, 400mM ethylamine hydrochloride, 300mM magnesium chloride hexahydrate, 150mM sodium hexametaphosphate, 5mM adenosine triphosphate (ATP) to the 100mL reaction flask in sequence, add water to dissolve the final volume to 50mL, The pH of the reaction was adjusted to 7.0 with 5M sodium hydroxide solution, and 250 mg of gamma-glutamyl methylamine synthase and 200 mg of phosphokinase were added. The reaction was carried out at 37°C under mechanical stirring. During the reaction, 2M sodium hydroxide solution was used to maintain the reaction pH at 7.0. After the reaction for 1 h, 250 mg of γ-glutamyl methylamine synthase and 200 mg of phosphokinase were added. After 24 hours of reaction, sampling was carried out for HPLC analysis, and the detection method was the same as that in Example 1.

[0050] The results showed that the conversion rate was 80% and the ee value was ...

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Abstract

The invention relates to the technical field of bioengineering and in particular relates to a synthetic method of L-theanine. The method comprises the following step of synthesizing L-theanine by taking gamma-glutamyl-methylamine synthetase and phosphokinase as catalysts and L-sodium glutamate and ethylamine hydrochloride as substrates. The method can be used for producing a lot of cloned expression proteins as catalysts by taking L-sodium glutamate and ethylamine hydrochloride which are relatively low in price as the initial raw materials, and has the advantages of being mild in reaction condition, strong in specificity, high in reaction conversion rate, simple and convenient to operate, low in requirement on reaction equipment, small in environmental pollution and the like.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for synthesizing L-theanine, in particular to a method for catalyzing ATP by using microbial enzymes and using sodium L-glutamate and ethylamine hydrochloride as substrates to couple the enzyme A method for synthesizing L-theanine in a regeneration system. Background technique [0002] L-Theanine (C 7 H 14 O 3 N 2 , also known as γ-glutamine ethylamine) is a special amino acid contained in tea, which has a variety of uses: used in ordinary food to improve food flavor, strengthen nutrition, etc.; in health food for sedation and relaxation, Improve sleep, anti-fatigue, improve learning and memory ability, etc. It has been used in the field of medicine to lower blood pressure, prevent cancer, prevent Alzheimer's disease, control depression, etc. Therefore, L-theanine has a wide range of needs in beverages, functional foods, and functional medicines. [0003] At...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P13/04
CPCC12P13/04
Inventor 祝俊李元刘珊
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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