Method for efficiently synthesizing L-theanine through recombined corynebacterium crenatum

A technology of corynebacterium bacilli and theanine, applied in the fields of genetic engineering and enzyme engineering

Pending Publication Date: 2016-11-09
JIANGNAN UNIV
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Problems solved by technology

Corynebacterium blunt-toothed SDNN403 is obtained through multi-stage mutagenesis screening and is suitable for high-yield safe strains of various ...

Method used

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  • Method for efficiently synthesizing L-theanine through recombined corynebacterium crenatum
  • Method for efficiently synthesizing L-theanine through recombined corynebacterium crenatum
  • Method for efficiently synthesizing L-theanine through recombined corynebacterium crenatum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Cloning of γ-glutamyl transpeptidase gene and construction of its expression vector

[0038] Using the pXMJ19 vector as a template, PrimerSTAR HS high-fidelity enzyme was selected, and two pairs of primers Nde I R, TacM-F, Nco I F, and tacM-R were used to amplify the tac-lacIq-Nde I fragment and the Nco I-oripUC-tac fragment respectively. The sizes of the gene fragments are about 2700bp and 2100bp respectively, and the overlapping sequence of the two fragments is ATTAATCATCG TGTGGTACCAT (The underlined part is the mutated site of the tac promoter and its mutated sequence). The above two DNA fragments were recovered from the gel and prepared for the next round of PCR. The PCR system includes dNTP, Pfu enzyme and its buffer, and the above two PCR products are added to make them serve as templates and primers (the total amount is set to 1 μL, and the dosage ratio can be adjusted according to the concentration of the PCR product). At a lower annealing temperatur...

Embodiment 2

[0041] Example 2: Construction of C. glutamicum SDNN403 recombinant bacteria and analysis of GGT protein expression

[0042] The recombinant plasmids pXMJ19-△sp ggt, pXMJ19-ggt, pXMJ19-tacM were transformed into C. glutamicum SDNN403 competent, and the positive recombinants C. glutamicum SDNN403 / pXMJ19-△sp ggt, C. glutamicum SDNN403 / pXMJ19-ggt were obtained by plasmid validation and screening , C. glutamicum SDNN403 / pXMJ19-tacM-ggt. Inoculate it in basal medium, cultivate overnight at 30°C, transfer to high-arginine-producing medium with 10% inoculum the next day, and add IPTG to a final concentration of 0.8mM in mid-logarithmic phase to induce expression , cultivated for 96h. After the culture, the supernatant was collected as the extracellular enzyme solution and stored at 4°C. The broken cell pellet was also reserved for subsequent analysis. The experimental results showed that only intracellular protein bands were observed in recombinant bacteria C.glutamicum SDNN403 / pX...

Embodiment 3

[0043] Embodiment 3: Recombinant bacterial strain GGT enzyme activity assay

[0044]After the recombinant bacteria C. glutamicum SDNN403 / pXMJ19-△sp ggt, C. glutamicum SDNN403 / pXMJ19-ggt and C. glutamicum SDNN403 / pXMJ19-tacM-ggt were cultured in the arginine high-yield medium, the intracellular fragmentation The enzyme activity of GGT in the supernatant and supernatant of the supernatant is shown in Table 1. Since the protein content inside and outside the cell is not comparable, only absolute enzyme activity units are considered here. In the recombinant strain C. glutamicum SDNN403 / pXMJ19-△sp ggt, only 0.99 U / mL of intracellular GGT activity was detected. Enzyme activity was detected both inside and outside the cell in the recombinant strain C.glutamicum SDNN403 / pXMJ19-ggt, which were 0.27U / mL and 4.69U / mL respectively. They are 0.32U / mL and 10.31U / mL respectively. It can be seen from the enzyme activity data (Table 1) that in the recombinant bacteria C. glutamicum SDNN403 / ...

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Abstract

The invention discloses a method for efficiently synthesizing L-theanine through recombined corynebacterium crenatum, and belongs to the field of genetic engineering and enzyme engineering. According to the method, it is firstly verified that GGT signal peptide of a bacillus subtilis source can achieve secretory expression in a C.glutamicum system. Meanwhile, extracellular enzyme activity of recombinant bacteria C.glutamicum SDNN403/pXMJ19-tacM-ggt is about two times that of C.glutamicum SDNN403/pXMJ19-ggt, and it indicates that a tac Mpromoter is more beneficial for synthesis of GGT enzyme activity. Substrate flow and strategic high-yield L-theanine are adopted, a conversion system contains GGT with the concentration of 0.8 u/mL, the pH is 10, the temperature is 37 DEG C, and 22 mmol/L L-theanine and 66 mmol/L ethylamine are supplemented every other two hours from 0 h. The maximum theanine yield of 118 mmol is achieved when batch flow is added for 12 h, and the conversion rate is 92.8%. The secretory expression of gamma-glutamyltranspeptidase genes (ggt) of the B.subtilis source in C.glutamicum SDNN403 is achieved for the first time, and the highest yield of L-theanine reported currently and synthesized through recombined C.glutamicum is obtained by adding the substrate in batch flow.

Description

technical field [0001] The invention discloses a method for expressing gamma-glutamyl transpeptidase protein by recombinant Corynebacterium blunt-tooth, and then using the protease to catalyze L-glutamine and ethylamine to generate L-theanine, which belongs to the field of genetic engineering and enzyme engineering. technical background [0002] L-theanine, whose chemical name is N-ethyl-γ-L-glutamine, is a free amino acid that almost only exists in tea plants, and determines the flavor and quality of tea. Theanine has a variety of important physiological functions: relaxation and decompression; blood pressure lowering; inhibition of excitement caused by caffeine; improvement of learning ability; anti-tumor; weight control, etc. As early as 1985, the U.S. Food and Drug Administration (FDA) has certified L-theanine as a generally recognized safe substance (GRAS), and there is no specific dosage limit for use in food. In view of the above-mentioned many physiological function...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P13/04C12R1/15
CPCC12N9/1044C12P13/04C12Y203/02013
Inventor 饶志明杨套伟和斐张显徐美娟
Owner JIANGNAN UNIV
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