Method for synthesizing L-theanine with enzyme

An enzymatic synthesis, theanine technology, applied in the field of L-theanine synthesis, can solve the problems of harsh conditions, long fermentation time, many side reactions, etc., and achieve high conversion rate, high conversion rate and simple reaction Effect

Inactive Publication Date: 2008-09-24
NANJING UNIV OF TECH
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  • Abstract
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Problems solved by technology

The preparation of L-theanine by chemical synthesis is the reaction of glutamic acid derivatives with ethylamine or ethylamine compounds, which involves the protection and deprotection of glutamic acid. The reaction route is complicated, the conditions are harsh, and there are many side reactions. And it is easy to produce racemates, and a large amount of organic solvents will pollute the environment
The fermentation method has the

Method used

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  • Method for synthesizing L-theanine with enzyme

Examples

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Example Embodiment

[0031] Example 1:

[0032] Take 15g of sucrose, 30g of corn syrup, 15g of peptone, K 2 HPO 4 15g, MgSO 4 0.5g, pH 7.5 and tap water are added to make 1L liquid culture medium. According to this formula, 3.5L is prepared and put into a 5L fermenter, sterilized by 0.1MPa high pressure steam for 25 minutes. At the same time, aliquot 50mL medium to 500mL, add 8 layers of gauze, wrap it in kraft paper, and sterilize it with the fermentation tank for later use.

[0033] Take out the bacteria Bacillus subtillis NX-2 (preservation number: CGMCCNo.0833) stored in the refrigerator, and then connect it to a fresh slant medium (slant medium (g / L): peptone 10, beef extract 3. On NaCl 5, agar 20), activate the culture for 24 hours, and insert the seed medium (seed medium (g / L): glucose 15, corn steep liquor 10, peptone 10, K 2 HPO 4 2. MgSO 4 0.25) Incubate with shaking at 33°C and 220r / min for 20 hours. At the end of the cultivation, it was connected to the fermentor, and the fermentation was ...

Example Embodiment

[0035] Example 2:

[0036] Weigh 7.3g (0.1mol) L-glutamine, 4.2g sodium bicarbonate dissolved in 50mL water, add 50mL copper sulfate pentahydrate through a constant flow pump, and control the reaction temperature at about 60°C. After the addition is complete, the reaction is continued at room temperature for about 2 hours, and then filtered, washed, and dried to obtain the L-glutamine-copper(II) complex.

Example Embodiment

[0037] Example 3:

[0038]Take 10.6mg (0.03mmol) of L-glutamine-copper(II) complex obtained in Example 2 in 2.5mL Tris-HCl buffer (pH 9, 0.1mol / L), add 2.5mL 400mmol / L Ethylamine was added to 0.8 mL of the γ-glutamyl transpeptidase obtained in Example 1, and the mixture was uniformly placed in a water bath for reaction for 2 hours. The reaction temperature was 37°C. After the reaction, the reaction solution was mixed with 1mol / L hydrochloric acid (volume is 10% of the reaction solution), and then analyzed and detected by high performance liquid chromatography system (chromatographic separation conditions: chromatographic column: DIONEX1Acclaim1205μC18; detection wavelength is 200nm; mobile phase is 95 % Water (containing 1‰ trifluoroacetic acid), 5% acetonitrile; flow rate: 1 mL / min), the conversion rate to glutamine is 71%.

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Abstract

The invention discloses an enzymatic preparation method of L-theanine by utilizing L-glutamine-Copper (II) complex as donor, which comprises that adopting chemical process to synthesize L-glutamine-Copper (II) complex and Gamma-glutamyltranspeptidase is added into the mixed aqueous solution of containing substrate of L-glutamine-Copper (II) complex and ethylamine for reaction. The method has the advantages of avoiding auto-transpeptidation problem, greatly promoting the conversion rate of donor, realizing the simple operation and the mild reaction condition as well as the low cost.

Description

technical field [0001] The present invention relates to a synthesis method of L-theanine, in particular to a method of using L-glutamine-copper (II) complex as a glutamyl donor and using γ-glutamyl transpeptidase to catalyze the synthesis of L-theanine -Theanine method. Background technique [0002] Theanine is an amide compound, which is a characteristic amino acid of tea and one of the main taste substances of tea. It exists in large quantities in tea trees and is a non-protein free amino acid. As an amino acid, theanine also has D-type and L-type, but the naturally occurring theanine is all L-type. In addition to flavoring, L-theanine also has many physiological functions: calming and relaxing nerves; enhancing learning and memory; lowering blood pressure; anti-tumor and improving the efficacy of anticancer drugs; protecting brain nerves; weight loss; Anti-fatigue effect. Therefore, L-theanine can be used in various industries such as pharmaceuticals, health products, ...

Claims

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Application Information

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IPC IPC(8): C12P13/04
Inventor 姚忠邓海霞周治吴明刚徐虹韦萍
Owner NANJING UNIV OF TECH
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