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Enterobacter aerogen and application thereof

A technology of Enterobacter aerogenes and strains, applied in bacteria, microorganisms, biochemical equipment and methods, etc., can solve the problems of many by-products, high adult body, low synthesis rate, etc.

Active Publication Date: 2010-05-05
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Traditional cadaverine is chemically synthesized using lysine, the synthesis rate is low, the by-products are many, and the adult body is high

Method used

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  • Enterobacter aerogen and application thereof
  • Enterobacter aerogen and application thereof
  • Enterobacter aerogen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Screening and preservation of strains

[0019] Medium

[0020] Intestinal bacteria enrichment broth medium: peptone 10g, glucose 5g, dipotassium hydrogen phosphate 2g, sodium dihydrogen phosphate 8g, distilled water 1000mL, pH value 7.2~7.4, sterilize at 115℃ for 15min;

[0021] Lower medium: peptone 5g, yeast extract 5g, beef extract 5g, sodium chloride 2.5g, glucose 0.5g, Tween 1g, magnesium sulfate 0.4g, manganese sulfate 0.03g, dipotassium hydrogen phosphate 2g, triammonium citrate 2g , Calcium carbonate 0.1g, ferrous sulfate 0.04g, vitamin B 1 0.01g, 0.05g pyridoxal phosphate, 18g agar. Putrescine, 5 grams each, 1000ml distilled water. pH5.0 (autoclaved at 115°C for 15 minutes);

[0022] Upper medium: 0.06g bromocresol purple, 20g agar, 1000ml distilled water, pH5.0 (sterilized at 121°C for 10min);

[0023] VRBDA agar medium: yeast extract 3g, peptone 7g, bile salt 1.5g, sodium chloride 5g, lactose 10g, neutral red 0.03g, crystal violet 0.002g, agar 15g, distilled water 1...

Embodiment 2

[0030] Genetic testing of Enterobacter aerogenes xlcad002

[0031] Primer for detecting lysine decarboxylase gene

[0032] CAD1-f: 5‘-TTYGAYWCNGCNTGGGTNCCNTAYAC-3’;

[0033] CAD1-r: 5‘-CCRTGDATRTCNGTYTCRAANCCNGG-3’;

[0034] Detection method:

[0035] 25μl reaction system includes: GoTaq Green Master Mix 12.5μl, each primer 1.0μl, DNA template 2μl, deionized double distilled water 8.5μl.

[0036] PCR amplification program: 94°C pre-denaturation for 5 minutes, 30 cycles including: 95°C denaturation for 30 seconds, 53°C annealing for 30 seconds, 72°C extension for 2 minutes, and finally 72°C extension for 7 minutes, and cooling to 4°C.

[0037] With the specific primers CAD1-f and CAD1-r for detecting lysine decarboxylase, a 1098bp gene fragment can be successfully amplified. figure 2 In, lane 1 is the gene fragment amplified in this study, lane n is the negative control. It can be seen that there is a lysine decarboxylase gene in Enterobacter aerogenes.

Embodiment 3

[0039] Method for preparing high-concentration cadaverine culture solution

[0040] Intestinal bacteria enrichment broth medium (same as Example 1);

[0041] Preparation

[0042] Pick colonies from the slope and inoculate them in a test tube containing A medium. Let stand for 24 hours at 37°C. The inoculation amount in the test tube is 10 5 cfu / ml. After repeated activation of the same medium for five times; inoculate the last activated bacterial solution into medium B and culture at 37°C for 4 days, the inoculum amount is 10 6 cfu / ml; Centrifuge the cultured bacterial solution at 10,000 rpm for 10 minutes, and take the supernatant to obtain a high-concentration cadaverine solution.

[0043] The A medium is an enterobacteria enrichment broth with 0.1% lysine; the B liquid medium is an enterobacteria enriched meat with 0.005% pyridoxal phosphate and 0.1% lysine Broth medium.

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Abstract

The invention relates to an enterobacter aerogen with the collection number of CGMCC No.3163. A bacterial colony is cultured on a VRBDA agar culture medium for 24 hours at the temperature of 30 DEG C and is a pink round bacterial colony; the bacterial colony is non-transparent, straight, peritrichous and Gram-negative and is in facultative anaerobe; in addition, the bacterial colony generates acid and gas when fermenting glucose; the VP test proves that the bacterial colony is gelatine hydrolysis positive and is rod-like under a common microscope. When being applied, the bacterial colony is inoculated into a test tube filled with culture medium A to stand at the temperature of 37 DEG C to be cultured for 24 hours; after being repeatedly activated five times, bacteria solution is inoculated into a culture medium B to stand at the temperature of 37 DEG C to be cultured for four days; the bacteria solution is decentralized at 1000 rpm for 10 minutes; supernate is extracted to obtain high concentration liquid; the culture medium A is enterobacteria enrichment broth added with 0.1% of lysine, and the culture medium B is enterobacteria enrichment broth added with 0.005% of phosphopyridoxal and 0.1% of lysine.

Description

Technical field [0001] The invention relates to a strain of microorganism and its application, and belongs to the field of biotechnology. Background technique [0002] The chemical name of cadaverine is 1,5-pentanediamine, which can be used in organic synthesis, biochemical reagents and pharmaceutical intermediates. At present, there is a large demand for cadaverine and its derivatives in my country, but no domestic enterprise can produce qualified Products imported from abroad have high prices. [0003] Traditional cadaverine is chemically synthesized using lysine, with low synthesis rate, many by-products and high adult body. [0004] Cadaverine is produced through microbial metabolism (lysine is decarboxylated to produce cadaverine under the action of the lysine decarboxylase produced by microorganisms) to obtain a high-concentration cadaverine culture solution, and then separate cadaverine, which can reduce the production cost and Energy consumption has also become the main subj...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P13/00C12R1/01
Inventor 徐幸莲卢士玲周光宏刘登勇舒蕊华
Owner NANJING AGRICULTURAL UNIVERSITY
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