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Recombinant corynebacterium crenatum capable of producing agmatine, and application thereof

A technology of Corynebacterium bluff and agmatine, applied in the biological field, can solve the problems of hindering the industrialization process of agmatine and high cost, and achieve the effects of low cost, few by-products and high output

Pending Publication Date: 2020-09-29
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the chemical synthesis method, the production of agmatine by the enzymatic conversion method has the advantages of simple steps, less by-products, and environmental protection. The market price of acid is as high as 50,000 to 80,000 yuan / ton. Therefore, the enzyme conversion method has the disadvantage of high cost, which greatly hinders the industrialization process of using the enzyme conversion method to produce agmatine

Method used

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  • Recombinant corynebacterium crenatum capable of producing agmatine, and application thereof
  • Recombinant corynebacterium crenatum capable of producing agmatine, and application thereof
  • Recombinant corynebacterium crenatum capable of producing agmatine, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: Construction of recombinant Corynebacterium blunt tooth

[0036] Specific steps are as follows:

[0037] (1) Use the genome of Escherichia coli (Escherichia coli) BL21 as a template, and use F and R as primers to carry out PCR amplification to obtain the gene speA encoding arginine decarboxylase as shown in SEQ ID No.2. ;

[0038] Wherein, the PCR amplification primers are:

[0039] F: 5'-ggtcgactctagaggatccAAAGGAGGAAAATCatgtctgacgacatgtctatg-3' (BamHI) (SEQ ID No. 3);

[0040] R: 5'-gccaaaacagccaagctgaattcttaGTGGTGGTGGTGGTGGTGctcatcttcaagataagtataaccgtacaaacctgcctcg-3' (EcoR 1) (SEQ ID No. 4);

[0041] PCR amplification conditions are: 95°C pre-denaturation, 5min; 95°C denaturation, 30s, 55°C annealing, 30s, 72°C extension, 90s, 30 cycles; 72°C final extension 5min;

[0042] The PCR amplification system is: template 1 μL, upstream and downstream primers 1 μL, sterilized double distilled water 22 μL, 2×Phanta MaxMaster Mix 25 μL.

[0043] (2) the gene...

Embodiment 2

[0045] Embodiment 2: the production of agmatine (whole cell transformation method)

[0046] Specific steps are as follows:

[0047] (1) With Corynebacterium crenatum SYPA5-5 as a control, a single colony of Corynebacterium crenatum SYPA5-5 was picked and inoculated into BHI liquid medium, and the recombinant blunt bacteria obtained in Example 1 A single colony of C. crenatum SYPA5-5 / pXMJ19-speA was inoculated to contain 10 μg·mL -1 In the BHI liquid medium of chloramphenicol, the temperature was 30°C and the rotation speed was 180rpm, and the shaker was cultured for 24 hours to obtain seed solutions 1-2; the seed solution 1 was inoculated with 2% (v / v) inoculum Into the BHI liquid medium, inoculate the seed liquid 2 with a 2% (v / v) inoculation amount to contain 10 μg·mL -1 In the BHI liquid medium of chloramphenicol, cultured on a shaker at a temperature of 30°C and a rotation speed of 180rpm for 10h, then added IPTG with a final concentration of 0.1mM to the culture medium,...

Embodiment 3

[0050] Embodiment 3: the production of agmatine (biological fermentation method+shaking flask)

[0051] Specific steps are as follows:

[0052] A single colony of the recombinant Corynebacterium crenatum SYPA5-5 / pXMJ19-speA obtained in Example 1 was picked and inoculated into the seed medium, and cultured on a shaker at a temperature of 30°C and a rotation speed of 180rpm for 24h to obtain Seed liquid: inoculate the seed liquid into the fermentation medium with a 10% (v / v) inoculum amount, and after cultivating on a shaker at a temperature of 30°C and a rotation speed of 220rpm for 24h, add a final concentration of 0.1 to the medium. mM IPTG was used to induce fermentation for 72 hours at a temperature of 30° C. and a rotation speed of 220 rpm to obtain a fermentation broth.

[0053] Detect the contents of L-arginine and agmatine in the fermentation broth, and the test results are: the content of agmatine in the fermentation broth obtained by C.crenatum SYPA5-5 / pXMJ19-speA fe...

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Abstract

The invention discloses a recombinant corynebacterium crenatum capable of producing agmatine, and application thereof, and belongs to the technical field of biology. The invention provides a recombinant corynebacterium crenatum C.crenatum SYPA5-5 / pXMJ19-speA; the recombinant corynebacterium crenatum is inoculated into a fermentation culture medium containing glucose to perform shake-flask fermentation for 72 h; therefore, the yield of agmatine in fermentation liquid is up to 9.7 g / L; simultaneously, the content of a by-product, namely L-arginine, is only 0.23 g / L; the recombinant corynebacterium crenatum is inoculated into the fermentation culture medium containing glucose to perform fermentor fermentation for 72 h; therefore, the yield of agmatine in the fermentation liquid is up to 37.44g / L; simultaneously, the content of the by-product, namely L-arginine, is only 0.76 g / L; and furthermore, L-arginine does not need to be added in the whole fermentation process.

Description

technical field [0001] The invention relates to a recombinant corynebacterium bacillus that can produce agmatine and an application thereof, belonging to the field of biotechnology. Background technique [0002] Agmatine is a derivative of arginine. Studies have shown that agmatine has biological activities such as lowering blood sugar, lowering blood pressure, diuresis, anti-inflammation, anti-depression, and inhibiting cell proliferation. The antagonism of (NMDA) receptor is strong and long-lasting, and it has the withdrawal effect on animal morphine dependence, and it is a kind of detoxification drug with great development value. Therefore, agmatine has a very broad market in the field of medicine. [0003] At present, the industry mainly uses two methods of chemical synthesis and enzymatic conversion to produce agmatine. Among them, the chemical synthesis method generally uses compounds such as 1,4-butanediamine, diethyl adipate, 1,4-dibromobutane and potassium phthal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P13/00C12R1/15
CPCC12N9/88C12P13/001C12Y401/01019
Inventor 徐美娟饶志明王怡许家钰杨套伟张显
Owner JIANGNAN UNIV
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