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L-theanine enzymatic transformation preparation method

A technology of theanine enzymatic method and glutamic acid, which is applied in the field of enzymatic conversion and preparation of L-theanine, achieves the effects of high substrate conversion rate, mild reaction conditions, and reduced production costs

Active Publication Date: 2009-10-21
ZHEJIANG TIANRUI CHEM
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  • Claims
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AI Technical Summary

Problems solved by technology

[0021] So far, using γ-glutamyl transpeptidase from microbial sources or genetic engineering sources as a catalyst, with L-glutamic acid-γ-alkyl ester or L-glutamylhydrazide and ethylamine as substrates, the enzyme The method for preparing L-theanine has not yet been reported in the literature

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 4

[0035] L-theanine enzymatic transformation preparation method, its specific steps are as follows:

[0036] 1. Add 17 g of the genetically engineered bacteria GGT (containing the recombinant plasmid pET28a-GGT) thallus obtained by centrifuging 1000 mL of fermentation broth into 1000 mL of transformation liquid, which contains 80 g of L-glutamic acid-γ-butyl ester, 120 g of ethylamine, 20mL of 1% polyethylene glycol octylphenyl ether, 10mL of 1mol / L magnesium chloride, 30% hydrochloric acid to adjust the pH to 10, and react at 35°C for 30h. The concentration (g / mL) of L-theanine in the reaction solution was 4.88%, and the molar conversion rate of the substrate L-glutamic acid-γ-butyl ester reached 71%.

[0037] 2. Centrifuge the reaction solution to remove bacteria, adjust the pH of the supernatant to 3 with 30% hydrochloric acid, heat up the acidification solution to 70-80°C, add activated carbon for decolorization, and pass the decolorization solution through a 732-type cation...

Embodiment 5

[0039] L-theanine enzymatic transformation preparation method, its specific steps are as follows:

[0040] 1. Add 23 g of Bacillus subtilis NX-2 cells obtained by centrifuging 1000 mL of fermentation broth into 1000 mL of transformation liquid, which contains 40 g of L-glutamyl hydrazide, 90 g of ethylamine, 20 mL of 0.5% Tween-80, 10 mL of 1mol / L potassium chloride, 30% hydrochloric acid to adjust the pH to 10, react at 45°C for 22h. The concentration (g / mL) of L-theanine in the reaction solution was 3.22%, and the molar conversion rate of the substrate L-glutamylhydrazide reached 74%.

[0041] 2. Centrifuge the reaction solution to remove bacteria, adjust the pH of the supernatant to 3 with 30% hydrochloric acid, heat up the acidification solution to 70-80°C, add activated carbon for decolorization, and pass the decolorization solution through a 732-type cation exchange resin column for adsorption. Elute the L-theanine saturated adsorption column with 3% ammonia water, col...

Embodiment 6

[0043] L-theanine enzymatic transformation preparation method, its specific steps are as follows:

[0044] 1. Add 20 g of Bacillus subtilis SK11.004 cells obtained by centrifuging 1000 mL of fermentation broth into 1000 mL of transformation liquid, which contains 60 g of L-glutamine, 150 g of ethylamine, and 10 mL of 1% hexadecyltrimethyl Ammonium bromide, 10mL 1mol / L sodium chloride, 30% hydrochloric acid to adjust the pH to 10, react at 40°C for 28h. The concentration (g / mL) of L-theanine in the reaction solution was 4.67%, and the molar conversion rate of the substrate L-glutamylhydrazide reached 72%.

[0045] 2. Centrifuge the reaction solution to remove bacteria, adjust the pH of the supernatant to 3 with 30% hydrochloric acid, heat up the acidification solution to 70-80°C, add activated carbon for decolorization, and pass the decolorization solution through a 732-type cation exchange resin column for adsorption. Elute the L-theanine saturated adsorption column with 3% a...

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Abstract

The invention belongs to the field of biotechnology, in particular relating to L-theanine enzymatic transformation preparation method. The preparation method takes L-glutamic acid-gamma-alkyl ester or L-glutamyl hydrazide and ethamine as raw materials, mixes bacteria cell containing gamma-glutamyltranspeptidase or enzyme extracting solution with L-glutamic acid-gamma-alkyl ester aqueous solution or the mixed aqueous solution of L-glutamyl hydrazide and ethamine, carries out enzymatic reaction under 25-60 DEG C, and separates the converted product by isoelectric point crystallization or by combining isoelectric point crystallization with ion exchange resin to obtain L-theanine with high purity. The method takes L-glutamic acid-gamma-alkyl ester or L-glutamyl hydrazide as raw material and has the advantages of low price of raw materials, convenient operation, short time of transformation and low preparation cost.

Description

1. Technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an enzymatic conversion preparation method of L-theanine. 2. Background technology [0002] L-Theanine (L-Theanine) is a natural amino acid present in tea, its structural formula is: CH 3 CH 2 NHCOCH 2 CH 2 CH(NH 2 )COOH. It is the main free amino acid and main taste substance in tea. Because L-theanine has many physiological functions such as lowering blood pressure, relaxing nerves, losing weight, anti-fatigue, improving immunity, etc., it is widely used in the fields of food, health care products and medicines. [0003] According to current literature reports, the preparation method of L-theanine mainly contains following three kinds: [0004] 1. Extracted from tea leaves [0005] The content of L-theanine in tea is relatively rich. In this method, tea leaves are steeped in boiling water, and then L-theanine is extracted by organic solvent extraction and io...

Claims

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Application Information

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IPC IPC(8): C12P13/04C12R1/125C12R1/19
Inventor 焦庆才张飞刘均忠熊吉滨刘茜
Owner ZHEJIANG TIANRUI CHEM
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