Method for preparing theanine by using species coupling ATP regenerative technology

A technology for preparing theanine and strains, which is applied to the preparation of theanine and the field of preparing theanine by using interspecies coupling ATP, can solve the problems of no selection, no theanine, and no clear concept, and achieves the goal of secondary The effect of less product, high production capacity and stable production process

Inactive Publication Date: 2011-07-27
GUANGDONG PHARMA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 1. There are reports in Japan on the production of theanine by co-cultivation technology. In essence, baker's yeast is used as the ATP regeneration bacteria, and Pseudomonas is the enzyme-producing strain, which catalyzes the transformation of theanine into γ-glutamyl in production. The enzyme of the transfer reaction is glutamine synthetase produced by Pseudomonas, but the literature report did not clearly propose the concept of interspecies coupling ATP regeneration technology
[0006] 2. There are domestic and foreign reports on products produced by interspecies coupling ATP regeneration technology, but there is no report on the production of theanine using this technology
[0007] 3. There are foreign reports that Bacillus stearothermophilus is the best ATP regeneration strain, but there is no report that the bacteria is used to prepare theanine
[0008] 4. There are reports at home and abroad that γ-glutamyl transferase is used for the production of theanine, but the γ-glutamyl transferase produced by Escherichia coli is selected to produce theanine, and the one produced by Bacillus licheniformis is not selected. Research report on production of theanine by γ-glutamyltransferase

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] ① Inoculate the slant of the enzyme-producing strain Bacillus licheniformis at 30°C for 48 hours, transplant the seed culture (30ml / bottle) at 30°C, 250 rpm for 24 hours, inoculate the fermentation culture (30ml / bottle) at 30°C, 250 rpm Cultivate for 24 hours to obtain enzyme-producing fermented liquid, fermented liquid 1000ml → 4000 revs / min, 15 minutes, obtain thalline 10 grams (gamma-glutamyl transferase activity 9u / ml). The bacterium was embedded with carrageenan to prepare γ-glutamyl transferase-immobilized cells (50 g) for later use.

[0027] ② ATP regeneration strain (Bacillus stearothermophilus)→activation culture→transformation to produce FDP→FDP transformation culture medium (56mgFDP / ml, cell content 1.5%) for later use.

[0028] ③ Mix 50 g of immobilized cells, 10 ml of FDP transformation medium, and 50 ml of substrate (glutamine 0.35 mol / L, ethylamine 2.0 mol / L), adjust the pH value to 10.5→30°C, and culture at 180 rpm for 48 Hours of conversion to produce ...

Embodiment 2

[0031] ① Inoculate the slant of the enzyme-producing strain Bacillus licheniformis at 30°C for 48 hours, transplant the seed culture (30ml / bottle) at 30°C, 250 rpm for 24 hours, inoculate the fermentation culture (30ml / bottle) at 30°C, 250 rpm Cultivate for 24 hours to obtain enzyme-producing fermented liquid, fermented liquid 1000ml→4000 rpm, 15 minutes, obtain thalline 10 grams (gamma-glutamyl transferase activity 8u / ml) for subsequent use.

[0032] ② ATP regeneration strain (Bacillus stearothermophilus)→activation culture→transformation to produce FDP→FDP transformation culture medium (53mgFDP / ml, cell content of 1.5%) for later use.

[0033]③Mix 10 grams of bacterial cells, 10 ml of FDP transformation culture medium, and 50 ml of substrate (glutamine 0.35 mol / L, ethylamine 2.0 mol / L), adjust the pH value to 10.5→30°C, and culture at 180 rpm for 48 Hours of conversion to produce theanine→theanine conversion liquid (theanine content 39mg / ml). The conversion rate of glutamin...

Embodiment 3

[0037] ① Inoculate the slant of the enzyme-producing strain Bacillus licheniformis at 30°C for 48 hours, transplant the seed culture (30ml / bottle) at 30°C, 250 rpm for 24 hours, inoculate the fermentation culture (30ml / bottle) at 30°C, 250 rpm Cultivate for 24 hours to obtain enzyme-producing fermentation liquid (gamma-glutamyl transferase activity 2u / ml) for subsequent use.

[0038] ② ATP regeneration strain (Bacillus stearothermophilus)→activation culture→transformation to produce FDP→FDP transformation culture medium (53mgFDP / ml, cell content of 1.5%) for later use.

[0039] ③Mix 20ml of enzyme-producing fermentation broth, 10ml of FDP transformation culture broth, and 50ml of substrate (glutamine 0.35mol / L, ethylamine 2.0mol / L), adjust the pH value to 10.5→30°C, and culture at 180 rpm for 48 Hours of conversion to produce theanine→theanine conversion solution (theanine content 35mg / ml). The conversion rate of glutamine to theanine was 56%.

[0040] ④Theanine extraction a...

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PUM

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Abstract

The invention discloses a method for preparing a theanine by an interspecific coupling ATP regenerative technology comprising the following steps: preparing gamma-glutamyltransferase immobilized cells by using bacillus licheniformis, preparing 1,6-diphosphate conversion culture solution by using bacillus stearothermophilus; preparing the theanine conversion solution by mixing the gamma-glutamyltransferase immobilized cells, 1,6-diphosphate conversion culture solution and substrates which are glutamine (glutamic acid) and ethylamine; obtaining the theanine by extracting and refining the theanine conversion liquid. The technical scheme provided by the invention is a fine new technical scheme for theanine preparation by coupling the ATP regenerative technology and an enzyme conversion technology, a technology capable of promoting the ATP consumed enzyme reaction smooth, also a technology suitable for the theanine production by the microorganism conversion method and has a wide application prospect in the food, medical treatment and the enzyme engineering field.

Description

technical field [0001] The invention relates to a method for preparing theanine, in particular to a method for preparing theanine by coupling ATP between species, and belongs to the technical fields of enzyme engineering and microbial fermentation. Background technique [0002] ATP regeneration technology (ATP regeneration technology) is a kind of technology that closely links the ATP generation process with the enzyme reaction that consumes ATP, so as to promote the smooth progress of the enzyme reaction that consumes ATP. At present, ATP regeneration technology is in the exploration stage and close to the practical application stage, and is facing a breakthrough. It is the bottleneck technology of intermediate product fermentation and enzyme engineering, and is the key technology to realize the production of many biologically active substances with microbial cells (or enzymes). Its in-depth research aims to regulate the biochemical process of ATP regeneration under special...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P13/04C12R1/07C12R1/10
Inventor 胡立勇张文军邓祖军傅锦坚孟佩佩刘冬英
Owner GUANGDONG PHARMA UNIV
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