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Method for producing L-ornithine by whole cell transformation of recombinant Corynebacterium crenatum

A technology of ornithine and recombinant bacteria, which is applied in the direction of recombinant DNA technology, bacteria, and the use of vectors to introduce foreign genetic materials, etc., can solve the problems of long fermentation cycle, high technology requirements, low fermentation yield, etc., and achieve product specificity Strong, high conversion efficiency

Inactive Publication Date: 2015-11-25
JIANGNAN UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The production of ornithine by fermentation can use relatively simple carbon sources, such as glucose, etc., and the cost is very low, which is suitable for industrial development, but the fermentation cycle is generally relatively long, and the yield of fermentation is low, which requires more separation in the later stage. high technical requirements

Method used

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  • Method for producing L-ornithine by whole cell transformation of recombinant Corynebacterium crenatum

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Experimental program
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Effect test

Embodiment 1

[0036] Example 1: Arginase Primer Design

[0037] According to the argI gene sequence in the whole genome nucleic acid sequence of Bacilluscereus in NCBI, the PCR primers P1 and P2 of the arginase gene were designed.

[0038] F: 5'-ACCCG AAGCTT ATGAAAAAAGAAATCTCAGTTATTGG-3'(HindIII)

[0039] R: 5'-ACCG GGATCC TTATTTTAGTTTTTCACCGAATAAAG-3' (BamHI)

Embodiment 2

[0040] Embodiment 2: the cloning of arginase gene

[0041] Using the total DNA of Bacilluscereus as a template, use the primers provided above for PCR amplification. The amplification conditions are: 94°C pre-denaturation, 5min, one cycle; 94°C denaturation, 1min, 56°C annealing, 1min, 72°C extension, 45s, 35 cycles; final extension at 72°C for 10 min. PCR amplification system: template 1 μL, upstream and downstream primers 0.4 μL, dNTPMix 4 μL, 10×ExTaqBuffer 5 μL, sterilized double distilled water 37 μL, ExTaq DNA polymerase 1 μL. The PCR product was purified and recovered using a gel recovery kit, and the concentration of the recovered product was checked by electrophoresis. The recovered product was stored in a 1.5ml centrifuge tube and stored in a -20°C refrigerator for later use. The recovered product was ligated with pMD18-TVector, and the ligated product was transformed into E.coilJM109, and the transformed product was coated on an LB plate containing ampicillin, cul...

Embodiment 3

[0042] Embodiment 3: Construction of recombinant plasmid pXMJ19-argI

[0043] Extract the plasmids pMD18-T-argI and pXMJ19 stored in E.coliJM109, and perform double enzyme digestion with BamHI and HindIII, respectively, and ligate after recovering with a gel recovery kit. Ligation system: 7 μL of digested product of the target gene, pXMJ19 Digest product 1 μL, T4 DNA ligase buffer 1 μL, T4 DNA ligase 1 μL, ligate overnight at 16°C. The connected recombinant plasmid pXMJ9-argI was transformed into competent E.coilJM109, and positive colonies were picked with LB chloramphenicol resistance medium. The plasmid was extracted after overnight culture on a shaker at 37°C and named pXMJ9-argI. After the enzyme digestion was verified to be correct, glycerol was added to a final concentration of 15%-20% (w / v), and stored in a -70°C refrigerator for later use.

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Abstract

Arginase from Bacillus cereus is successfully expressed in C. crenatum SDNN403 through shuttle plasmid pXMJ19 between Escherichia coli and Corynebacterium crenatum. Results of enzymatic determination show that an original bacterium has no activity of the arginase; enzyme activity of the arginase of a recombinant engineering bacterium is up to 24.3 U / mg. By using a whole-cell body of the recombinant engineering bacterium as a biocatalyst and the L-ornithine as a substrate, on the basis of enzymatic properties of the arginase, an optimal reaction temperature being 40 DEG C and pH being 9.0 are determined; 200ml of LBG cultivated recombinant bacterium is resuspended in 200ml of substrate buffer solution; the L-ornithine is produced by means of transformation under optimal transformation conditions; after 8h, 149.2g / L L-ornithine is acquired; molar conversion rate of substrate L-ornithine is up to 99.5%. The L-ornithine produced by the method has the advantages of high efficiency, high specificity, low energy consumption and the like.

Description

technical field [0001] The invention relates to the cloning and expression of the arginase gene to obtain a recombinant engineering bacterium, and simultaneously applies the engineering bacterium to the production of L-ornithine, which belongs to the field of bioengineering and biotechnology. technical background [0002] L-arginase (E.C.3.5.3.1) is a key enzyme in the urea cycle, which catalyzes the formation of L-ornithine and urea from L-arginine. Arginase is widely found in various animals, plants and In microorganisms, arginases from microorganisms such as Bacillus brevis, Bacillus subtilis and Bacillusthuringiensis have been well studied. [0003] Ornithine is a basic amino acid, easily soluble in water and ethanol, slightly soluble in organic solvents such as ether. Ornithine accepts two molecules of NH 4 + and a molecule of CO 2 One molecule of L-arginine is formed. L-arginine can also be hydrolyzed into ornithine and urea under the action of arginase. [0004]...

Claims

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Application Information

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IPC IPC(8): C12N15/77C12N1/21C12P13/10
Inventor 饶志明王梅洲徐美娟张显杨套伟
Owner JIANGNAN UNIV
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