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Method for producing L-ornithine hydrochloride by genetic engineering bacteria

A technology of ornithine hydrochloride and genetically engineered bacteria, which is applied in the field of production of L-ornithine hydrochloride and can solve problems such as difficulty in filtration

Active Publication Date: 2013-05-08
WUHAN GRAND HOYO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the method of chemically hydrolyzing L-arginine to generate L-ornithine often causes L-ornithine racemization, the method for preparing L-ornithine by weak base hydrolysis of L-arginine can reduce the degree of racemization, However, it is necessary to use sulfuric acid to precipitate weak alkali metal ions, which will encounter the problem of difficulty in filtration, and will produce certain solid waste

Method used

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  • Method for producing L-ornithine hydrochloride by genetic engineering bacteria
  • Method for producing L-ornithine hydrochloride by genetic engineering bacteria
  • Method for producing L-ornithine hydrochloride by genetic engineering bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0181] Design and synthesize a pair of TA cloning primers

[0182] Log in to GenBanK and obtain the arginase gene of Bacillus Subtilis 168 strain through Gene ID: 937760 ( arg The complete coding sequence of Bsub), using Primerer 5.0 software to design a pair of PCR primers, p1, p2, for amplifying the DNA sequence of Bacillus subtilis arginase gene, and performing TA ligation with pBAD / Thio-TOPO vector. Primers were synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd.

[0183] P1: 5′-GAAATGGATAAAACGATTTCGGTTAT-3′, a total of 26 bp.

[0184] P2: 5′-GGTCAGCAGCTTCTTCCCTAACA-3′, a total of 23 bp.

Embodiment 2

[0186] Extraction and determination of Bacillus subtilis genomic DNA

[0187] Genomic DNA of Bacillus subtilis was extracted by the improved method of Palva I et al.

[0188] (1). Pick a single bacterium colony cultured on the plate of Bacillus subtilis (preservation number CCTCC No. 93009), inoculate it into a test tube with 10 ml of beef extract and peptone liquid medium, and culture it with shaking at 32° C. for 14 hours.

[0189] (2). Centrifuge at 5000rpm for 10min to obtain bacterial pellet, wash once with STE, centrifuge again, and resuspend the bacterial cell in 4ml TE solution.

[0190] (3). Add 8 μl of 50 mg / ml lysozyme solution (final concentration is 100 μg / ml), and incubate at 37°C for 20 minutes. Then add RNase (10mg / ml) 10μl to a final concentration of 25μg / ml, then add 10% SDS to dissolve 0.5ml, and keep warm at 37℃ for 30min. Add proteinase K (20mg / ml) 10μl, the final concentration is 50μg / ml, keep at 37℃ for 60min.

[0191] (4). Then add an equal volume of...

Embodiment 3

[0200] PCR Amplification of Arginase Gene of Bacillus subtilis

[0201] 1). Prepare 50 μl reaction system.

[0202] Template DNA, 2μl (50ng); 10 X PCR Buf, 5μl; 50mMdNTPs, 0.5μl;

[0203] Primers p1 and p2, each 1μl (100ng); Taq DNA polymerase (1 unit / μl), 1μl; add sterile pure water to a volume of 50μl.

[0204] The program of PCR reaction was: pre-denaturation at 94°C for 4min, denaturation at 94°C for 1min, annealing at 56°C for 30sec, extension at 72°C for 1.5min; a total of 30 cycles, and finally extension at 72°C for 10min. After the reaction was completed, temporarily store in ice until use.

[0205] 2). Take 2μl of the amplified product and load it on 1.2% agarose gel electrophoresis to detect a 0.9KB DNA fragment. The DNA band has a clear outline and is suitable for direct TOPO-TA cloning reaction.

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Abstract

The invention discloses a method for producing L-ornithine hydrochloride by TOP10 / PBAD / Thio-TOPO-Arg-Bsub colibacillus genetic engineering bacteria. The strain can express and generate excess bacillus subtilis arginase, and efficiently converts L-arginine into L-ornithine.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to the application of genetically engineered bacteria, in particular to a method for producing L-ornithine hydrochloride by using Escherichia coli TOP10 / PBAD / Thio-TOPO-Arg-Bsub genetically engineered bacteria. Background technique [0002] 1. Physiological functions and health care value of L-ornithine: [0003] L-Ornithine is a non-protein amino acid, and it is a precursor for organisms to synthesize protein amino acids such as arginine and proline. L-Ornithine can be converted into polyamines such as putrescine, cadaverine, spermidine, and spermine in vivo, and polyamines can promote the growth of biological tissues. Positively charged polyamines can combine with negatively charged DNA and RNA to promote DNA transcription and RNA translation in organisms; polyamines can combine with proteins or phospholipids on the cell membrane to maintain the stability of the membrane. [0004] L-orn...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P13/10C12N1/21C12N15/60C12N15/70C12R1/19
Inventor 王炯王君英刘爱福易清明
Owner WUHAN GRAND HOYO
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