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Construction of arginase production engineering bacteria and application of arginase production engineering bacteria in production of L-ornithine

A technology of arginase and arginase gene, which is applied in the direction of bacteria, the use of vectors to introduce foreign genetic material, recombinant DNA technology, etc., can solve the problems of complex components, low yield, and difficult separation, so as to reduce production costs , The reaction speed is improved, which is beneficial to the absorption effect

Inactive Publication Date: 2014-07-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cost of raw materials for producing ornithine by fermentation is low, but the yield is not high. At the same time, due to the complexity of the components in the fermentation broth, subsequent separation is relatively difficult, and the average fermentation cycle is as long as 50 hours or more.

Method used

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  • Construction of arginase production engineering bacteria and application of arginase production engineering bacteria in production of L-ornithine
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  • Construction of arginase production engineering bacteria and application of arginase production engineering bacteria in production of L-ornithine

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Experimental program
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Effect test

Embodiment 1

[0047] Construction of Genetic Engineering Bacteria Containing Arginase

[0048] (1) The arginase gene from Bacillus caldovelox (DSM411) (GenBank accession number: U48226 .1) Perform PCR amplification: Add LAtaq enzyme to the system, pre-denature at 94°C for 3 minutes, denature at 94°C for 30 seconds, anneal at 55°C for 30 seconds, extend at 72°C for 1.2 minutes, 30 cycles, and finally extend at 72°C for 10 minutes;

[0049] (2) Digest the target gene and expression vector pET28a at 37°C for 2 hours with restriction endonucleases Nco I and HindIII;

[0050] (3) Use T4 ligase to ligate the target gene and plasmids pET20b and pET28a after digestion and gel recovery at 16°C for 10 h;

[0051] (4) Introduce the constructed expression plasmid into E.coli BL21(DE3), and culture it on the LB plate containing kanamycin for 12h;

[0052] (5) Perform PCR and enzyme digestion verification on the colony grown on the plate, carry out sequencing verification on the plasmid containing the ...

Embodiment 2

[0054] Conversion of L-arginine to L-ornithine by arginase obtained through expression and secretion

[0055] (1) Insert the genetically engineered bacteria of construction into LB slant medium and cultivate for 12h;

[0056] (2) Put a ring of slanted seeds into the LB medium and cultivate for 6 hours;

[0057] (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, collecting the thalli after 6h, and washing the thalline twice with sterile normal saline;

[0058] (4) Put the thalli into the transformation solution for transformation, and the transformation conditions are as follows: the substrate arginine concentration is 160g / L, the transformation temperature is 50-70°C, the transformation time is 2-4h, and the shaker speed is 150rpm;

[0059] (5) Take an appropriate amount of conversion solution for detection and analysis

Embodiment 3

[0061] The transformation solution was centrifuged at 10,000 rpm for 2 minutes, the supernatant was collected, and a standard solution was prepared using L-ornithine hydrochloride as a standard. The moderately diluted supernatant and standard solution were respectively derivatized with 2,4-dinitrofluorobenzene, filtered through a 0.22 μm microporous membrane, and verified whether the product was L-ornithine by liquid chromatography-mass spectrometry. Determination of L-ornithine content by high performance liquid chromatography.

[0062] The relative molecular mass of the fermentation product and the standard sample is the same as measured by liquid chromatography-mass spectrometry (see image 3 ), it can be proved that the conversion product is L-ornithine.

[0063] The content of L-ornithine in the conversion solution was measured by high performance liquid chromatography, and it can be seen that after 4 hours, the output of L-ornithine reached 120.1 g / L, and the conversion...

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Abstract

The invention describes a construction method of arginase production engineering bacteria and application of the arginase production engineering bacteria in production of L-ornithine by transformation of L-arginine, and belongs to the technical field of biological engineering. According to the method, by means of molecular biology, thermophilic bacteria Bacillus caldovelox (DSM411) is cloned to obtain arginase gene, a constructed expression plasmid is transfected into E.coli BL21 (DE3), and expression vector high-copy recombine ant arginase-containing engineering bacteria pET28a-ARG is obtained by kanamycin resistance plate screening. The enzyme can still show high activity after induced expression in E.coli. The bacteria obtained by fermentation can be directly used for transformation without breaking, in the condition of 60 DEG C, the engineering bacteria has higher permeability, and is more conducive to the substrate uptake and product release, the reaction speed is greatly improved, the transformation period is only 2-4h, the ornithine yield can reach 120.1g / L, and the transformation rate is 98.9%.

Description

technical field [0001] The invention relates to a construction method of an arginase-producing engineering bacterium and how to use the bacterium to produce L-ornithine, belonging to the field of bioengineering. Background technique [0002] 1. The medical value and physiological function of L-ornithine [0003] L-Ornithine is one of the ubiquitous amino acids in organisms. Ornithine is a non-protein amino acid and is a precursor for the synthesis of protein amino acids such as proline and arginine. In animals, ornithine is mainly involved in the urea cycle (see figure 1 ). [0004] L-ornithine is an important metabolic compound in cells, and it has a detoxifying effect on ammonia accumulated in the body. L-ornithine can stimulate the pituitary gland to secrete growth hormone, promote protein synthesis and catabolism of sugar and fat. In addition, L-ornithine can increase the synthesis of polyvinylamine, promote cell proliferation, and play a certain role in improving im...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12P13/10
Inventor 刘立明宋伟王鸿江
Owner JIANGNAN UNIV
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