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Arginine decarboxylase and application thereof

A technology of arginine decarboxylase and arginine, applied in the direction of application, enzyme, lyase, etc., can solve the problems of many steps, high cost, unsuitable for large-scale industrial production, etc., and achieve the effect of clear ingredients

Inactive Publication Date: 2016-08-17
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This route has many steps, low yield, high cost, and is not suitable for large-scale industrial production

Method used

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  • Arginine decarboxylase and application thereof
  • Arginine decarboxylase and application thereof
  • Arginine decarboxylase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: Containing the construction of arginine decarboxylase genetically engineered bacteria

[0038] Construct genetically engineered bacteria containing arginine decarboxylase as follows:

[0039] (1) Use primers 1 whose sequences are respectively shown in SEQ ID NO.3 and SEQ ID NO.4 by means of molecular biology:

[0040] 5'-CGGAATTCATGAATGATTGGTCTATTGATGAT-3' and Primer 2:

[0041] 5'-CCGCTCGAGTTAAGAAAAATCTTCTAAATAA-3' The arginine decarboxylase gene (amino acid sequence as shown in SEQ ID NO.2, nucleotide sequence as shown in SEQ ID NO.1) from Shewanella putrefaciens was amplified by PCR Addition: add rTaq enzyme to the system, pre-denature at 95°C for 5 minutes, denature at 94°C for 30 seconds, anneal at 55°C for 30 seconds, extend at 72°C for 2.0 minutes, 30 cycles, and finally extend at 72°C for 10 minutes;

[0042] (2) Digest the target gene and expression vector pET28a at 37°C for 4 hours with restriction enzymes EcoR I and Xho I;

[0043] (3) Ligate t...

Embodiment 2

[0046] Example 2: Induced expression of arginine deiminase

[0047] Induce the expression of genetically engineered bacteria as follows:

[0048] (1) Insert the genetically engineered bacteria of construction into LB slant medium and cultivate for 12h;

[0049] (2) Put a ring of slanted seeds into the LB medium and cultivate for 6 hours;

[0050] (3) Insert the seed solution into the LB fermentation medium and cultivate to OD 600 0.6, induced at three temperatures of 16, 25 and 37°C, adding IPTG with a final concentration of 0.05, 0.1, 0.2 and 0.4mmol / L, respectively, and collecting the bacteria after 4, 8, 10 and 16 hours of induction, and sterile physiological Bacteria were washed with salt water.

Embodiment 3

[0051] Embodiment 3: the impact of conversion conditions on the output of agmatine

[0052] Cultivate the genetically engineered bacterium constructed in Example 1, and induce it to express arginine decarboxylase, then collect the genetically engineered bacterium, and wash the thalline twice with sterile physiological saline;

[0053] The protein was purified using a nickel column, and the protein concentration was detected with a Bradford kit; different transformation systems were used to determine the content of agmatine in the transformation solution by high performance liquid chromatography.

[0054] (1) The transformation system is: pure enzyme 5.0×10 4 U / L, the substrate arginine concentration is 140g / L, the transformation temperature is 37°C, and the transformation time is 24h; under the transformation conditions, the yield of agmatine can reach 71.9g / L, and the average production intensity of the whole transformation process is 3.0g / (L·h), the conversion rate reached ...

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Abstract

The invention discloses arginine decarboxylase and application thereof and belongs to the technical field of biological engineering. Shewanella putrefaciens is cloned by adopting genome database mining and homologous sequence alignment and other virtual screening means and combining with a molecular biological means to obtain an arginine decarboxylase gene. The molecular biological means, process optimization and the like are adopted, the enzyme production capacity of arginine decarboxylase production strains is improved, and a platform for efficiently producing gamatine through biological catalysis is established by optimizing a catalytic system. Crude enzyme liquid obtained through fermentation is purified and then is converted, a reaction system is clear in composition, and extraction and purification of follow-up products are promoted. Under the condition of 37 DEG C, the decarboxylase has high activity, meanwhile is beneficial to the growth of escherichia coli of host cells, and at the temperature, the reaction speed is improved greatly, a conversion period should be 24 hours, the gamatine yield can be up to 61-71 g / L, and the conversion rate can be up to 68-82%.

Description

technical field [0001] The invention relates to an arginine decarboxylase and its application, belonging to the technical field of bioengineering. Background technique [0002] Agmatine (Agmatine), also known as herring spermine, molecular formula C 5 h 14 N 4 , an endogenous clonidine displacing substance (CDS), is an agonist of imidazoline receptor α 2 - Non-catecholamine ligands for adrenergic receptors. It exists in various tissues and cells of organisms, plays an important role in the metabolism of organisms, and has various biological functions. In the cardiovascular system, agmatine can reduce heart rate, mean arterial pressure, left ventricular pressure, peripheral vascular resistance index, etc.; in the urinary system, agmatine can increase the glomerular filtration rate and absolute proximal impulse absorption; In the intestinal tract, agmatine can increase the secretion of gastric acid and pepsin, make the mucosa thinner, and worsen the mucosal lesions caused...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/60C12N9/88C12P13/00
CPCC12N9/88C12P13/001C12Y401/01019
Inventor 刘立明孙安然宋伟刘佳罗秋玲
Owner JIANGNAN UNIV
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