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Method for producing L-theanine by supplementing material

A theanine and feeding technology, applied in the biological field, can solve the problems of less development of enzyme species, low wild enzyme activity, limited industrial application process, etc., and achieve a short reaction period, high raw material conversion rate and low production cost. Effect

Inactive Publication Date: 2015-02-25
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the existing research, the development of γ-glutamyl transpeptidase (γ-GGT) enzymes is less, and the activity of wild enzymes is low, and the effect on the production and application of L-The needs to be improved. However, there are few shortcomings such as less research on the lower reaction, and these shortcomings limit its industrial application process.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Substrate preparation:

[0030] The concentration of L-Gln was 200mmol / L, ethylamine hydrochloride was added according to the ratio of 1:10, and the pH was adjusted to 10.0 with buffer.

[0031] Enzymatic production process:

[0032] After the substrate solution is prepared, adjust the temperature to 37°C, add 6 units of γ-GGTase per milliliter of the reaction solution, then add 200 mmol L-Gln every 2 hours, and add ethylamine hydrochloride regularly to keep the excess. A total of 14 hours of reaction;

[0033]The analysis of the L-The adopts high performance liquid chromatography. Chromatographic conditions: Waters 600HPLC chromatograph, Waters autosampler, detection system: Agilent 1200, chromatographic column: Eclipse XDB-C185μm (4.6mm×150mm); mobile phase A is 4.52g of anhydrous sodium acetate, add 1L of deionized water, then add 225 μL triethylamine, adjust the pH to 7.2±0.05 with acetic acid, and then add 5 mL tetrahydrofuran. Phase B is 4.52g of anhydrous sod...

Embodiment 2

[0035] Substrate preparation:

[0036] Same as Example 1

[0037] Enzymatic production process:

[0038] After the substrate solution is prepared, adjust the temperature to 37°C, add 6 units of γ-GGTase per milliliter of the reaction solution, then add 150 mmol L-Gln every 2 hours, and regularly add ethylamine hydrochloride to keep the excess. A total of 14 hours of reaction;

[0039] L-theanine detection method:

[0040] Same as Example 1

[0041] The total concentration of substrate L-Gln is 950mmol / L, and the total conversion rate of L-The can reach 63.7%.

Embodiment 3

[0043] Substrate preparation:

[0044] Same as Example 1

[0045] Enzymatic production process:

[0046] After the substrate solution is prepared, adjust the temperature to 37°C, add 6 units of γ-GGTase per milliliter of the reaction solution, then add 100 mmol L-Gln every 2 hours, and regularly add ethylamine hydrochloride to keep the excess. A total of 16 hours of reaction;

[0047] L-theanine detection method:

[0048] Same as Example 1

[0049] The total concentration of substrate L-Gln is 900mmol / L, and the total conversion rate of L-The can reach 67.8%.

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Abstract

The invention relates to a method for producing L-theanine by supplementing a material, belonging to the technical field of biology and solving the technical problems of enhancing the transformation rate of L-theanine in the presence of a high-concentration substrate and reducing the production cost by providing a process for supplementing the material at constant speed and supplementing the material at variable speed. The technical scheme is as follows: the method comprises the following steps of: producing L-theanine by adopting gamma-GGTase derived from a bacillus subtilis source; preparing L-glutamine and ethylamine hydrochloride according to a proportion of 1:5 to 1:15; predetermining temperature at 30-45 DEG C, regulating the pH value to be 9.0-11.0, then adding a 2-20 unit amount of gamma-GGTase to each milliliter of reaction liquid, supplementing a certain quantity of substrates every 2 hours, and reacting for 10-20 hours to obtain L-theanine with higher concentration.

Description

technical field [0001] The invention relates to a method for producing L-theanine by supplementing materials, and belongs to the field of biotechnology. Background technique [0002] L-theanine (L-The), also known as N-ethyl-γ-glutamine (5-N-ethyl-γ-glutamine) or γ-glutamyl-L-ethylamine (γ-glutamyl- L-ethylamine), first isolated from green tea by Japanese scientists in 1949, is a unique free amino acid in tea and the main flavor substance of tea. K. Susaoka et al. successfully isolated the enzyme from the seed root of tea for the first time, and proved that it has high affinity to L-The, ethylamine and ATP, but the enzyme has poor stability and is easily inactivated in vitro. [0003] In recent years, according to more and more research reports, L-theanine has important physiological functions such as sedation and decompression, improving cognition, enhancing immunity and preventing tumors. At the same time, it is widely used as a food additive in beverages, candies, biscui...

Claims

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Application Information

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IPC IPC(8): C12P13/04C12R1/125
Inventor 吴敬陈晟傅嘉懿
Owner JIANGNAN UNIV
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