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Strain for producing arginine deiminase and application thereof

An arginine deiminase and strain technology, which is applied in the field of arginine deiminase production by fermentation of Pseudomonas mutans CGMCC.2039, can solve the problems such as the production of ADI by fermentation of Pseudomonas mutans.

Inactive Publication Date: 2008-02-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on Pseudomonas plecoglossicida as a human pathogen, and there is no report on the production of ADI by Pseudomonas plecoglossicida

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Screening of ADI-producing strains

[0041] 5 soil samples, 2 plant samples, and 2 water samples were taken from Wuxi Huishan, Xihui Park, and the Wuxi section of the Beijing-Hangzhou Grand Canal, for a total of 9 samples. Quickly screen the strains within 12 hours after sampling. The sample was inoculated in the enrichment medium, 30°C, 160rpm, cultured for 24hrs, diluted, spread on a screening plate, 30°C, cultured for 72hrs, and colonies with red color changing circles were selected. A total of 54 strains with red color-changing circles were picked out. Select 18 colonies with large color change circles, inoculate them into a test tube containing 5mL of fermentation medium, and culture them at 30°C and 160rpm for 24 hours, and use the diacetylmonoxime-thiosemicarbazide method to measure the ADI enzyme activity of the strains screened out , to select strains with high enzyme activity.

[0042] Enrichment medium: L-arginine hydrochloride 15g / L, biotin 3mg / L, thiamin...

Embodiment 2

[0051] Identification of the SW-P1 strain

[0052] The bacterial strain SW-P1 obtained by screening in Example 1 is identified according to "Bergey's Bacteria Identification Handbook (Eighth Edition)" and "Common Bacteria System Identification Handbook" (see Table 2), and according to the refined molecular Chromosomal DNA was extracted according to the biological experiment guide method, and the 16S rDNA sequence of the strain was obtained by entrusting Dalian Bao Biotechnology Co., Ltd. , and homology comparison (Table 3). Based on the identification of morphology, physiological and biochemical characteristics, 16S rDNA sequence and phylogenetic analysis, the strain SW-P1 was identified as Pseudomonas metamorphosus, and it was proposed to be named Pseudomonas plecoglossicida SW-P1. Bacteria have been preserved in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures, and the preservation number is CGMCC No.2039.

[0053] Table 2 Com...

Embodiment 3

[0086] Enzyme Production by Fermentation of CGMCC 2039 Strain

[0087] Slope culture: According to the medium formula: peptone 2.5g / L, yeast extract 2.5g / L, beef extract 2.5g / L, NaCl 5g / L, agar 20g, pH7.0. Make a slant according to a conventional method, inoculate with the CGMCC 2039 strain obtained by screening in Example 1, and incubate at 30° C. for 24 hours.

[0088] Seed and fermentation medium formula: glucose 15g / L, yeast extract 3g / L, peptone 3g / L, L-arginine hydrochloride 5g / L, Na 2 HPO 4 12H 2 O12g / L, NaH 2 PO 4 2H 2 O3g / L, (NH 3 ) 2 HPO 4 2g / L, pH7.0;

[0089] The filling volume of the seed medium is 60ml / 250ml triangular flask. Connect a ring of cultured slant strains in a conical flask, 30°C, 190 rpm, and shake the flask for 14-18 hours.

[0090] Using a 5-liter fermenter, insert the cultivated shake flask seeds into the fermenter, the inoculum size is 2%, 30°C, the rotating speed is 300 rpm, the air volume is 1:2 volume / volume / min, and the defoamer is ...

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Abstract

The invention discloses a method to produce ADI strain by screening and the application of the method, pertaining to the field of bioengineering technology. The invention particularly relates to a rapid method to screen ADI strain and produce Pseudomonas plecoglossicida (CGMCC No.2039) of ADI, as well as a method to produce ADI by using the microorganism. Under an aerobic condition and an environment maintained at pH6.5-8.0, enzyme activity can achieve 1.6U / mL fermentation broth after 20-24h fermentation culture. ADI produced with such microorganism has very strong inhabitation agaisnt liver cancer cell strain (HepG2). ADI genes can be obtained from the chromosome DNA of such microorganism, and the gene order can be achieved.

Description

technical field [0001] A bacterial strain producing arginine deiminase and its application belong to the technical field of bioengineering. In particular, it relates to a strain and method for fermenting and producing arginine deiminase (ADI) with Pseudomonas plecoglossicida (Pseudomonas plecoglossicida) CGMCC. The application of the enzyme produced by the strain in medicine. Background technique [0002] Arginine deiminase (Arginine deiminase) is referred to as ADI. Many microorganisms use ADI to hydrolyze arginine to obtain energy. In the metabolic pathway of arginine deiminase, the first step is the hydrolysis of arginine by ADI to generate citrulline and ammonia. Arginine is a non-essential amino acid for humans. Normal nuclear tissue can be synthesized by citrulline under the action of the urea cycle enzymes arginine-succinate (ASS) synthase and arginine-succinate. Some human cancer cells do not express ASS and thus cannot synthesize arginine from citrulline. There...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/78C12N15/55A61K35/74C12R1/38
CPCA61K38/50C12N9/78A61P35/00
Inventor 孙志浩郑璞倪晔刘咏梅刘宇鹏
Owner JIANGNAN UNIV
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