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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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AI Technical Summary

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 following disadvantages: low product concentration and conversion rate, long fermentation time, difficult downstream separation, high equipment cost
[0006] However, the above enzymatic methods all use L-glutamine as a substrate, and there is a problem of rotating peptides, that is, because L-glutamine has an α-amino group, it can also be used as a γ-glutamyl receptor in the actual reaction to form glutamine Acylglutamine (Glu-Gln)

Method used

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  • Method for synthesizing L-theanine with enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Take 15g of sucrose, 30g of corn steep liquor, 15g of peptone, K 2 HPO 4 15g, MgSO 4 0.5g, pH 7.5, add tap water to make 1L liquid culture medium, prepare 3.5L according to this recipe, put it into a 5L fermenter and put it into a 0.1MPa high-pressure steam sterilization for 25 minutes for later use. At the same time, divide 50mL of medium into 500mL, add 8 layers of gauze, wrap it in kraft paper, and sterilize it together with the fermenter for later use.

[0033] After taking out the bacterial species preserved in the refrigerator—Bacillus subtillis NX-2 (preservation number is CGMCCNo.0833), connect it to fresh slant medium (slant medium (g / L): peptone 10, beef extract 3. On NaCl 5, agar 20), activate the culture for 24 hours, 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) Shaking culture at 33°C and 220r / min for 20 hours. At the end of the cultivation, it was connected to a fermenter, and ferm...

Embodiment 2

[0036] Weigh 7.3g (0.1mol) of L-glutamine, dissolve 4.2g of sodium bicarbonate in 50mL of water, add 50mL of copper sulfate pentahydrate through a constant flow pump, and control the reaction temperature at about 60°C. After feeding, continue to react at room temperature for about 2 hours, then filter, wash, and dry to obtain L-glutamine-copper (II) complex.

Embodiment 3

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