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Method for producing L-citrulline with high efficiency

A citrulline and high-efficiency technology, applied in the field of bioengineering, can solve problems such as insufficient conversion rate, low production intensity, and long conversion time, and achieve the effects of increasing reaction speed, reducing production cycle, and improving production efficiency

Active Publication Date: 2015-07-29
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the current production of L-citrulline by enzymatic conversion method has the problems of insufficient conversion rate, low yield, long conversion time and low production intensity.

Method used

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  • Method for producing L-citrulline with high efficiency
  • Method for producing L-citrulline with high efficiency
  • Method for producing L-citrulline with high efficiency

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Construction of genetically engineered bacteria containing arginine deiminase

[0041] Construct genetically engineered bacteria containing arginine deiminase as follows:

[0042] (1) Use primers 1 whose sequences are respectively shown in SEQ ID NO.3 and SEQ ID NO.4 by means of molecular biology:

[0043] 5'-CATGCCATGGCAATGAACAATGGAATTAATGTTAACTCAG-3' and Primer 2:

[0044]5'-CCGCTCGAGTTACAAATCTTCACGGCAAAGTGG-3'Arginine deiminase gene (amino acid sequence shown in SEQ ID NO.1, nucleotide sequence shown in SEQ ID NO.2) from Lactococcus lactis was subjected to PCR amplification : Add LAtaq enzyme to the system, pre-denature at 94°C for 3min, denature at 94°C for 30s, anneal at 55°C for 30s, extend at 72°C for 1.5min, 30 cycles, and finally extend at 72°C for 10min;

[0045] (2) Digest the target gene and expression vector pET28a with restriction enzymes NcoI and xhoI at 37°C for 2 hours;

[0046] (3) Use T4 ligase to ligate the target gene and plasmid pET28...

Embodiment 2

[0049] Embodiment 2: Induced expression of genetically engineered bacteria

[0050] Induce genetically engineered bacteria to express arginine deiminase as follows:

[0051] (1) Insert the genetically engineered bacteria of construction into LB slant medium and cultivate for 12h;

[0052] (2) Put a ring of slanted seeds into the LB medium and cultivate for 6 hours;

[0053] (3) Insert the seed solution into the LB fermentation medium and cultivate to OD 600 0.6, adding IPTG with a final concentration of 0.4mmol / L for induction, 6h later, the cells were collected, and the cells were washed with sterile normal saline.

Embodiment 3

[0054] Embodiment 3: Transform L-arginine to produce L-citrulline

[0055] Cultivate the genetically engineered bacterium constructed in Example 1, and induce it to express arginine deiminase, then collect the genetically engineered bacterium, and wash the thalline twice with sterile physiological saline;

[0056] Take 7g (wet weight) of the bacteria, crush it and put it into 1L of transformation solution for transformation. The transformation conditions are: the concentration of substrate arginine is 200g, the transformation temperature is 50°C, pH 7.2, and the transformation time is 8h;

[0057] Take an appropriate amount of transformation liquid, and use high performance liquid chromatography to determine the content of L-citrulline in the transformation liquid. The results show that the output of L-citrulline can reach 120.9g / L, and the production intensity can reach 15.1g·(L·h) -1 .

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Abstract

The invention discloses a method for producing L-citrulline with high efficiency, and belongs to the technical field of bioengineering. The method comprises the steps of cloning in lactic acid bacteria by molecular biology methods to obtain peptidylarginine deiminase gene, improving the enzyme producing ability of ADI production strains by molecular biology, process optimization and other methods, and building a platform for producing L-citrulline by high-efficiency biological catalysis by optimizing a catalytic system. The bacteria obtained after fermentation can be directly used for conversation without being broken, the engineering bacteria have higher permeability at 50 degrees centigrade, so the absorption of substrate and release of products are promoted further, the reaction speed is improved greatly, the conversation period is only 4-12h, the yield of L-citrulline can reach 150-180g / L, and the conversation rate is greater than 94%.

Description

technical field [0001] The invention relates to a method for efficiently producing L-citrulline, which belongs to the technical field of bioengineering. Background technique [0002] L-citrulline is one of the ubiquitous amino acids in organisms. It is a non-protein amino acid. L-citrulline can protect DNA and enzymes by resisting hydroxyl groups, and can protect DNA from oxidation reactions. The accumulation of citrulline can enhance the antioxidant effect of cells and protect cells from oxidation damage. The application of L-citrulline in medicine is mainly as an indicator of allogeneic rejection. In the treatment of diseases, the vasodilation effect of citrulline also plays an important role. In the body, citrulline can be converted from arginine, and this reaction releases endogenous NO, which can maintain normal blood pressure. In the diagnosis and treatment of rheumatoid arthritis (RA), the role of citrulline has also begun to receive people's attention. Studies ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/10C12N9/78C12N15/70
Inventor 刘立明刘佳宋伟
Owner JIANGNAN UNIV
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