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Escherichia coli strain for efficiently converting glutamine to synthesize L-theanine and application thereof

A technology of Escherichia coli and glutamine, which is applied to Escherichia coli strains that efficiently transform glutamine to synthesize L-theanine and its application field, and can solve problems such as restricting applications and screening for efficient transformation of theanine Escherichia coli strains

Active Publication Date: 2010-02-10
NANJING NORMAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Due to the difficulty of chiral separation of L-theanine through chemical synthesis, which restricts the application of this method in large-scale industrial production, the use of microbial fermentation has become an inevitable direction for large-scale industrial production , but it is very difficult to screen Escherichia coli strains that can efficiently transform theanine

Method used

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  • Escherichia coli strain for efficiently converting glutamine to synthesize L-theanine and application thereof
  • Escherichia coli strain for efficiently converting glutamine to synthesize L-theanine and application thereof
  • Escherichia coli strain for efficiently converting glutamine to synthesize L-theanine and application thereof

Examples

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Effect test

Embodiment 1

[0020] Example 1 strain screening

[0021] 23 Escherichia coli strains isolated from tea tree soil were inoculated into LB medium containing ethylamine (0.45M) for overnight culture.

[0022] After 24 hours of culture, the cultured E. coli strains were observed with a microscope, and it was found that the cell morphology of 3 strains of E. coli strains was normal and had a large number of cells, while the cell morphology of the other 20 strains was irregular and the number of cells was very large. few.

[0023] Add 400mM glutamine and 1M ethylamine to E. coli cells in the logarithmic growth phase for biotransformation. After 12 hours of further fermentation, most of the glutamine will be synthesized by L-theanine synthase in E. coli strains converted to theanine.

[0024] The results of theanine conversion by fermentation of the above three strains of Escherichia coli were analyzed by HPLC, and one of the strains of Escherichia coli had a conversion rate of glutamine exceedi...

Embodiment 2

[0027] (1) Inoculate the screened Escherichia coli Nanshi No. 1 in LB solid medium, containing 10g of peptone per liter, 5g of yeast extract, 10g of NaCl, 15g of agar, culture at 37°C for 12 hours, and inoculate a single colony in 10mL LB liquid medium, each liter of medium contains 10g of peptone, 5g of yeast extract, 10g of NaCl, dilute to 1 liter with deionized water, shake and culture at 37°C for 12h, then inoculate 1mL into 100mL of LB liquid medium, at 37°C Shake culture for 6~8h, OD 600 reach 2.0~2.2( figure 1 ), enzyme activity unit (Nanjing Jiancheng Biological Kit detection) OD 530 Reach 12000~14000( figure 2 ), collected by centrifugation for 15 minutes, and crushed under high pressure to obtain theanine synthase.

[0028] (2) Add 4000 units of theanine synthase per 0.6M / L glutamine and 70% (mass) ethylamine aqueous solution, after 40-48 hours of enzyme reaction at 25°C, it is found that it is sensitive to glutamine by HPLC Amide conversion was as high as 70%. ...

Embodiment 3

[0031] (1) Inoculate the screened Escherichia coli Nanshi No. 1 in LB solid medium, containing 10g of peptone per liter, 5g of yeast extract, 10g of NaCl, 15g of agar, culture at 37°C for 12 hours, and inoculate a single colony in 10mL Autonomous MB medium with Na per liter medium 2 HPO 4 15g, KH 2 PO 4 5g, NaCl 1g, NH 4 Cl 2g, glycerol 3ml, peptone 10g; culture with shaking at 37°C for 12h, then inoculate 1mL into 100mL autonomous MB liquid medium, culture with shaking at 37°C for 6-8h, OD 600 reach 2.1~2.3( image 3 ), enzyme activity unit (Nanjing Jiancheng Biological Kit detection) OD 530 Reach 15000~16000( Figure 4 ), centrifuged for 15 minutes to collect the thalline, and crushed under high pressure to obtain the gamma-glutamyl transferase synthesized by theanine

[0032] (2) Add 5000 units of theanine synthase per 0.6M / L glutamine and ethylamine aqueous solution, and after 40-48 hours of enzymatic reaction at 25°C, it is found that the conversion rate of gluta...

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Abstract

The invention relates to an Escherichia coli strain for biologically converting to synthesize theanine and an application thereof. The Escherichia coli strain for efficiently converting L- glutamine and ethylamine is Escherichia coli of CCTCC No. M 209166. The strain can prepare theanine synzyme in high yield, can efficiently convert the glutamine and the ethylamine into L-theanine and obtains success in the industrial production process. The converting ratio of the glutamine exceeds 80%.

Description

Technical field: [0001] The invention relates to an Escherichia coli strain for producing theanine through high-efficiency biotransformation and its application in industrial production of theanine. Background technique [0002] L-theanine is a unique amino acid in green tea. In the 1950s, Japanese scholars first extracted and refined theanine from tea leaves, and determined its chemical structure. Since then, many scholars have carried out a lot of research work on it. Generally speaking, the preparation methods of theanine mainly include plant cell tissue culture, chemical synthesis, microbial fermentation and ion exchange resin separation. A large number of studies have shown that: theanine has a variety of nutritional and health functions, calming the nerves and relaxing, promoting brain function, lowering blood pressure, anti-tumor and anti-fatigue effects. Theanine is an emerging food additive, which is safe and non-toxic, and has been widely used in medicine, health ...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P13/04C12R1/19
Inventor 殷志敏沈青红吕志祥王正才傅珒张正平
Owner NANJING NORMAL UNIVERSITY
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