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Synthetic method of L-theanine

A synthesis method, theanine technology, applied in the field of bioengineering, can solve the problems of unfavorable industrial production, complex reaction system and low efficiency

Active Publication Date: 2017-06-27
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 enzymatic method catalyzes the synthesis of L-theanine

[0042] Into a 50mL reaction bottle, sequentially add 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) , add water to dissolve so that the final volume is 10 mL, adjust the reaction pH to 7.0 with 5M sodium hydroxide solution, add 50 mg γ-glutamine synthetase and 40 mg 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 (5 mM, 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] Embodiment 2 enzymatic method catalyzes the synthesis of L-theanine

[0046] In the 100mL reaction bottle, add successively the final concentration of 200mM sodium glutamate, 200mM ethylamine hydrochloride, 150mM magnesium chloride hexahydrate, 75mM sodium hexametaphosphate, and 5mM adenosine triphosphate disodium (ATP), 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 γ-glutamine synthetase and 200 mg of phosphokinase were added. The reaction was carried out at 37° C. under mechanical stirring, and the reaction pH was maintained at 7.0 with 2M sodium hydroxide solution during the reaction. After reacting for 7 hours, samples were taken for HPLC analysis and the detection method was the same as in Example 1.

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

Embodiment 3

[0048] Embodiment 3 enzymatic method catalyzes the synthesis of L-theanine

[0049] In the 100mL reaction bottle, add successively the final concentration of 400mM sodium glutamate, 400mM ethylamine hydrochloride, 300mM magnesium chloride hexahydrate, 150mM sodium hexametaphosphate, 5mM adenosine disodium triphosphate (ATP), 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 γ-glutamine synthetase and 200 mg of phosphokinase were added. React at 37°C under mechanical stirring. During the reaction, use 2M sodium hydroxide solution to maintain the reaction pH at 7.0. After 1 hour of reaction, add 250mg of γ-glutamine synthetase and 200mg of phosphokinase. After reacting for 24 hours, samples were taken for HPLC analysis, and the detection method was the same as in Example 1.

[0050] The results showed that the conversion rate was 80%, and the ee value was >99%.

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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 microbial enzyme-catalyzed ATP coupling enzyme method with L-sodium glutamate and ethylamine hydrochloride as substrates Method for synthesizing L-theanine in a regenerative system. Background technique [0002] L-theanine (C 7 h 14 o 3 N 2 , also known as γ-glutamyl ethylamine) is a special amino acid contained in tea, which has many uses: it is used in ordinary food to improve food flavor and strengthen nutrition, etc.; in health food, it is used for calming and relaxing, Improve sleep, anti-fatigue, improve learning and memory ability, etc.; in the field of medicine, it has been used to lower blood pressure, prevent cancer, prevent Alzheimer's disease, control depression, etc. Therefore, L-theanine has a wide demand in beverages, functional foods, and functional medicines. [0003] At present, the production...

Claims

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Application Information

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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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