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Enterococcus faecalis and application thereof

A technology of Enterococcus faecium and colony, applied in bacteria, microorganisms, biochemical equipment and methods, etc., can solve the problems of large environmental pollution, low production efficiency, difficult to purify, etc., to reduce production costs, save energy, and tyramine capacity. strong effect

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] The traditional production of tyramine adopts chemical synthesis method, which causes great pollution to the environment, low production efficiency and difficult to purify

Method used

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  • Enterococcus faecalis and application thereof
  • Enterococcus faecalis and application thereof
  • Enterococcus faecalis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Screening and Preservation of Enterococcus faecium xltyr002

[0017] Medium:

[0018] MRS liquid medium is: peptone 10g, beef extract 10g, yeast extract 5g, diammonium hydrogen citrate 2g, glucose 20g, Tween 80 1mL, sodium acetate 5g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.5g, manganese sulfate 0.25 g, 1000 mL of distilled water, pH 6.2-6.4, sterilized at 115°C for 15 minutes.

[0019] The lower medium is: 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 18g, tyrosine 5g, 1000ml distilled water. pH 5.2, autoclaved at 115°C for 15 minutes.

[0020] The upper medium is: bromocresol violet 0.06g, agar 20g, 1000ml distilled water, pH 5.2, sterilized at 121°C for 10min.

[0021] MRS solid medium: add 1.8% aga...

Embodiment 2

[0028] Genetic testing of Enterococcus faecium xltyr002.

[0029] Primer

[0030] TD2: 5'-ACATAGTCAACCATRTTGAA-3';

[0031] TD5: 5'-CAAATGGAAGAAGAAGTAGG-3';

[0032] Detection method:

[0033] 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.

[0034] PCR amplification program: pre-denaturation at 94°C for 5 min, 35 cycles including: denaturation at 94°C for 45 s, annealing at 48°C for 45 s, extension at 72°C for 60 s, final extension at 72°C for 7 min, and cooling to 4°C.

[0035] Using the specific primers TD2 and TD5 that can amplify the tyrosine decarboxylase gene, a gene fragment of 1100bp can be amplified successfully. figure 2 Among them, lane 1 is the gene fragment amplified from the DNA of Enterococcus faecium. It can be seen that the gene of tyrosine decarboxylase exists in Enterococcus faecium.

Embodiment 3

[0037] Preparation method of high-concentration tyramide culture solution Pick colonies from the inclined plane and inoculate them into test tubes containing A medium, and culture them at 37°C for 24 hours, and the inoculum size in the test tubes is 10 5 cfu / ml. In the same medium, Enterococcus faecium was repeatedly activated five times and inoculated into B medium for 4 days at 37°C, with an inoculum size of 10 6 cfu / ml; Centrifuge the cultured bacterial solution at 10,000 rpm for 10 minutes, take the supernatant to obtain a high-concentration tyramine solution, and the tyramine content of the high-concentration tyramine solution is 4023.94 μg / ml.

[0038] Medium: MRS liquid medium (with embodiment 1); Described A medium is: add the MRS liquid medium of 0.1% tyrosine; Described B medium is: add 0.005% pyridoxal phosphate+ MRS broth with 0.1% tyrosine.

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Abstract

The invention relates to an enterococcus faecalis with the collection number of CGMCC No.3165. A bacterial colony is cultured on an MRS agar culture medium at the temperature of 37 DEG C for 24 hours to be gathered; the bacterial colony is white, oval, is in short chain and Gram-negative, generates acid when fermenting glucose and is elliptic under a common microscope. When being applied, the bacterial colony is selected from a slope to be 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, the finally activated bacteria solution is inoculated into a culture medium B to stand at the temperature of 37 DEG C to be cultured for four days; inoculation amount is 106cfu / ml; cultured bacteria solution is decentralized at 1000 rpm for 10 minutes; and supernate is extracted to obtain high concentration tyramine solution. The culture medium A is an MRS liquid culture medium added with 0.1% of tyrosine, and the culture medium B is an MRS liquid culture medium added with 0.005% of pyridoxal phosphate and 0.1% of tyrosine.

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 tyramine is p-hydroxy-β-phenylethylamine, which is an important intermediate in the synthesis of drugs and can be used as a biochemical reagent. At present, there is a large demand for tyramine 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 production of tyramine adopts chemical synthesis method, which causes great pollution to the environment, low production efficiency, and is difficult to purify. Using tyrosine as a precursor, using tyrosine decarboxylase produced by microorganisms to produce tyramine through microbial metabolism, obtaining high-concentration tyramine liquid, and then separating tyramine can reduce the production cost and energy consumption of tyramine,...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P13/00C12R1/01
Inventor 徐幸莲卢士玲周光宏刘登勇韩衍青
Owner NANJING AGRICULTURAL UNIVERSITY
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