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Method for biosynthesizing theanine by gene-engineering strain

A genetically engineered bacteria and biosynthesis technology, applied in the field of applied microorganisms, can solve the problems of high cost, low production rate, and inability to industrialize, and achieve the effects of easy control, high efficiency, and simple operation

Inactive Publication Date: 2009-06-03
TEA RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] However, the cost of extracting high-purity theanine from tea leaves is still very expensive. Although methods such as tea tree callus and suspension cell culture can be used to produce natural L-theanine, the production rate is high due to high operating costs. Low, not yet ready for industrialization

Method used

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  • Method for biosynthesizing theanine by gene-engineering strain
  • Method for biosynthesizing theanine by gene-engineering strain
  • Method for biosynthesizing theanine by gene-engineering strain

Examples

Experimental program
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Effect test

Embodiment 1

[0032] (1) The frozen-preserved strain (preservation number: CGMCC No.2714, containing the recombinant plasmid pET32a-GGT, see figure 1 ) was inoculated in LB medium containing Amp, and cultured at 37°C for about 16 hours to activate it. The activated strains were inserted into LB medium, and cultured in a shaking table at 37°C and 180r / min for about 12h to obtain seed liquid.

[0033] (2) Draw 0.5mL of seed fermentation liquid to contain the fermentation medium containing Amp (optimum fermentation medium glucose 10.39g / L, yeast extract 6.88g / L, K 2 HPO 4 7.10g / L, peptone 10g / L, NaCl10g / L, MgSO 4 ·7H 2 (01g / L) in the Erlenmeyer flask, 37 DEG C, 180r / min vibration culture 4h, then add 0.05mmol / L IPTG, 32 DEG C induce 4 hours, obtain the genetically engineered bacterium fermented liquid that contains γ-glutamyl transpeptidase.

[0034] (3) Centrifuge the fermented liquid of the genetically engineered bacteria obtained by fermentation at 8000r / min, remove the supernatant, was...

Embodiment 2

[0036] (1) Inoculate the frozen-preserved strain (preservation number: CGMCC No. 2714) into LB medium containing Amp, and culture it at 37° C. for about 16 hours to activate it. The activated strains were inserted into LB medium, and cultured in a shaking table at 37°C and 180r / min for about 12h to obtain seed liquid.

[0037] (2) Draw 0.5mL of seed fermentation liquid to contain the fermentation medium containing Amp (optimum fermentation medium glucose 10.39g / L, yeast extract 6.88g / L, K 2 HPO 4 7.10g / L, peptone 10g / L, NaCl10g / L, MgSO 4 ·7H 2 (01g / L) in the Erlenmeyer flask, 37 DEG C, 180r / min vibration culture 6.6h, then add 0.05mmol / L IPTG, induce 4 hours at 31.5 DEG C, obtain the genetically engineered bacterium fermented liquid that contains γ-glutamyl transpeptidase .

[0038] (3) Centrifuge the fermented liquid of the genetically engineered bacteria obtained by fermentation at 8000r / min, remove the supernatant, wash with sterile water and add theanine biosynthesis r...

Embodiment 3

[0039] The HPLC analysis of embodiment 3 synthetic theanine

[0040] After using the bacterial liquid for fermentation, the fermented liquid is used to determine the theanine content in the fermented liquid according to the following steps

[0041] (1) Mobile phase preparation

[0042] Mobile phase A: 1L of mobile phase A (60mM anhydrous sodium acetate, 1% N, N-dimethylformamide, adjusted to pH 6.4 with glacial acetic acid), mobile phase B: 1L of 50% acetonitrile.

[0043] (2) Pre-column derivatization reaction of theanine

[0044] ① Take the supernatant collected in 3.2.3 and adjust the pH to neutral with hydrochloric acid.

[0045] ②Take 100ul of the neutralized supernatant and put it into a 1.5ml Eppendorf centrifuge tube, then add 100ul of NaHCO3 (0.5M, pH9.0) and 100ul of DNFB (prepared in hexanenitrile) in sequence, and keep at 60°C Light reaction for 1 hour (note: shake frequently during the period, so that the sample is in full contact with the amino acid derivatizer ...

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Abstract

The invention provides a gene-engineering stain which contains recombinant plasmid pET32a-GGT with the plasmid size of 7563bp and the GGT segment size of 1743bp, and is called Escherichia coli; the preservation number of gene-engineering stain in Chinese microorganism strain preservation center is CGMCC No.2714; the gene-engineering stain has ampicillin resistance and carries out induced expression of Gama-glutamyltranspeptidase by Isoprophylthio-beta-D-galactoside. The method establishes the gene-engineering strain with theanine biosynthesis capability by cloning Gama-GGT in E.coli DH5Alpha, has simple operation, high efficiency, facilitates the control in the production process, and has high output and yield of theanine and excellent industrial application prospect.

Description

technical field [0001] The invention belongs to the field of applied microorganisms, and relates to a method for biosynthesizing theanine by genetic engineering bacteria. Background technique [0002] Theanine is a characteristic amino acid in tea and one of the taste substances of tea. It was discovered by Yajiro Sato in 1950 from the shoots of Gyokuro tea and named Theanine. From the discovery so far, it has not been found in other plants except for trace amounts detected in fungi, camellia, mushrooms, camellia oleifera, and red camellia. Theanine is also the free amino acid with the highest content in tea trees. In the shoots and leaves of tea trees, about 70% of the free amino acids are theanine. In recent years, in the study of tea health components, theanine is a more attractive compound. After its many physiological functions were discovered, how to synthesize and extract theanine has become a research topic of widespread concern. [0003] Theanine belongs to amide...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/54C12P13/02C12R1/19
Inventor 成浩王丽鸳周健陆文渊王贤波林智
Owner TEA RES INST CHINESE ACAD OF AGRI SCI