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Enterobacter aerogen and application thereof

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

Active Publication Date: 2012-06-06
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] culture medium

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

[0021] Lower layer 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, pyridoxal phosphate 0.05g, agar 18 grams. Putrescine, 5 grams each, 1000ml distilled water. pH 5.0 (autoclave at 115°C for 15 minutes);

[0022] Upper medium: bromocresol purple 0.06g, agar 20g, 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, ...

Embodiment 2

[0030] Gene detection of Enterobacter aerogenes xlcad002

[0031] Primers for detection of lysine decarboxylase gene

[0032] CAD1-f:5'-TTYGAYWCNGCNTGGGTNCCNTAYAC-3';

[0033] CAD1-r:5'-CCRTGDATRTCNGTYTCRAANCCNGG-3';

[0034] Detection method:

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

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

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

Embodiment 3

[0039] Preparation method of high-concentration cadaverine culture solution

[0040] Enterobacteria enrichment broth medium (with embodiment 1);

[0041] Preparation

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

[0043] The A medium is the enterobacteria enrichment broth medium adding 0.1% lysine; the B liquid medium is the enterobacteria enrichment meat adding 0.005% pyridoxal phosphate and 0.1% lysine Soup 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, belonging 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 there is no domestic enterprise that can produce qualified Products, imported from abroad are expensive. [0003] The traditional cadaverine is chemically synthesized by lysine, the synthesis rate is low, the by-products are many, and the adult body is high. [0004] Produce cadaverine through microbial metabolism (lysine is decarboxylated to produce cadaverine under the action of lysine decarboxylase produced by microorganisms), obtain high-concentration cadaverine culture solution, and then separate cadaverine, which can reduce the production cost and cost of cadaverine Ene...

Claims

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Application Information

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Patent Type & Authority Patents(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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