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Method for catalyzing to generate L-theanine by using recombinant Bacillus subtilis secreted gamma-glutamyltranspeptidase

A technology of glutamyl transpeptidase and recombinant vector, which is applied in the fields of genetic engineering and enzyme engineering, can solve problems such as hidden dangers of food additives, and achieve the effects of high raw material utilization, easy control, and simple process

Inactive Publication Date: 2015-03-11
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Escherichia coli, as an opportunistic pathogenic bacteria, has some hidden dangers in the production of food additives

Method used

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  • Method for catalyzing to generate L-theanine by using recombinant Bacillus subtilis secreted gamma-glutamyltranspeptidase
  • Method for catalyzing to generate L-theanine by using recombinant Bacillus subtilis secreted gamma-glutamyltranspeptidase

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Embodiment 1: Construction of recombinant B.subtilis 168 bacterial strain

[0028] Bacillus subtilis with γ-glutamyl transpeptidase activity in the laboratory was used as the starting strain, and its chromosome was used as a template to obtain the gene of the enzyme by means of PCR, which was connected to the cloning vector to achieve a large amount of amplification of the gene. A large number of amplified GGT gene fragments were purified and connected to the pMA5 vector. After successful verification, the model strain Bacillus subtilis 168 was transformed. Positive transformants were screened on the resistance plate, inoculated with shake flasks for fermentation, and the enzyme activity in the fermentation broth was measured by colorimetry, and the enzyme was purified for L-theanine conversion. The production of theanine was detected by HPLC, indicating that the construction was successful. A recombinant strain with the ability to secrete γ-glutamyl transpeptidase was ...

Embodiment 2

[0029] Embodiment 2: the enzyme activity assay of recombinant bacterial strain

[0030] Inoculate 1% of the inoculum into 50mL / 250mL optimized medium, take the cultured bacterium solution for 60h, and centrifuge at 10,000r / min for 30min at 4°C to collect the supernatant, which contains the target protein. The enzyme activity determination method is as follows: 1ml of the standard reaction system contains 50mM borate-sodium hydroxide (pH 10), 2.5mM γ-L-glutamyl-4-nitroaniline, 60mM diglyceride, 750mM NaCl and 10 μl of appropriately diluted enzyme solution. After reacting at 37°C for 10 minutes, 2ml of 3.5N acetic acid was added to terminate the reaction. The reaction system was not added with diglycyl dipeptide as a control, and the absorbance value was measured at 410nm. The difference in absorbance between the experimental group and the control group was the transpeptidase activity. Transpeptidase activity is defined as: under standard reaction conditions, the amount of enz...

Embodiment 3

[0031] Example 3: Purification of secreted enzyme liquid from recombinant strains and its use in catalytic production of theanine

[0032] Purification method: The supernatant was filtered through a 0.45 μm filter membrane, and Ni-NTA affinity chromatography was used to achieve one-step purification. The cleaning and elution procedures of the protein were carried out in accordance with the instructions.

[0033] L-theanine conversion method: use 80mM L-glutamine as the donor, 640mM ethylamine as the acceptor, adjust the pH to 10, add GGT to a concentration of 0.6U / ml, and react at 37°C for 5 hours, 10 % trichloroacetic acid to terminate the reaction. The production of theanine was detected by HPLC. The formation of 68.9mM L-theanine was detected, and the conversion rate could reach 86%.

[0034]

[0035]

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Abstract

The invention relates to a method for catalyzing L-glutamine and ethylamine to generate L-theanine by using recombinant Bacillus subtilis secreted gamma-glutamyltranspeptidase and belongs to the field of genetic engineering and enzyme engineering. The method provided by the invention is used for amplifying genes of gamma-glutamyltranspeptidase (GGT) in bacillus subtilis and cloning overexpression in bacillus subtilis 168. According to the method, a bacillus subtilis engineering strain capable of secreting gamma-glutamyltranspeptidase is established for the first time and the enzyme activity of the strain and the fermenting property of enzyme are researched: the supernatant enzyme activity of recombinant bacteria reaches 49.8 U / mg which is 20 times of that of an original strain. By using 80mML-glutamine as a donor and 640mMZ ethylamine as a receptor, the conversion ratio of L-theanine can reach over 86%. The method provided by the invention uses the safe strain, has the advantages of being high in conversion ratio, simple in production method and the like, and facilitates production of L-theanine produced by an industrial scaled enzymic method.

Description

technical field [0001] The invention discloses a method for catalyzing L-glutamine and ethylamine to generate L-theanine by using gamma-glutamyl converting enzyme secreted by recombinant Bacillus subtilis, belonging to the fields of genetic engineering and enzyme engineering. technical background [0002] L-theanine (N-ethyl-L-glutamic acid), a natural amino acid that exists in large amounts in tea leaves, was originally extracted from tea leaves by Sakato in the late 1940s. The results of in vitro studies show that intake of theanine can help relieve stress, improve cognitive ability, reduce the incidence of cancer and vascular diseases, and enhance the body's immunity. L-theanine has been certified as a safe food additive by the US Food and Drug Administration (FAD). Due to the many benefits of theanine appeal, its demand is increasing day by day. [0003] The production methods of L-theanine include: direct extraction from natural resources, chemical synthesis and biosy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N1/21C12N9/10C12P13/04C12R1/125
Inventor 饶志明杨套伟张显徐美娟贝纳和斐
Owner JIANGNAN UNIV
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