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Enterococcus faecalis and application thereof

A technology of Enterococcus faecalis and colony, applied in the directions of bacteria, microorganisms, biochemical equipment and methods, etc., can solve the problems of low production efficiency, large environmental pollution, difficult purification, etc., and achieve energy saving, production cost reduction, tyramine ability strong effect

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] 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 faecalis xltyr001

[0017] Medium:

[0018] MRS liquid medium is: peptone 10g, beef extract 10g, yeast extract 5g, diammonium hydrogen citrate 2g, glucose 20g, Tween 801mL, sodium acetate 5g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.5g, manganese sulfate 0.25g , distilled water 1000mL, pH 6.2-6.4, sterilized at 115°C for 15min.

[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 18 grams. Tyrosine 5 grams, 1000ml distilled water. pH 5.2, autoclave 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% ag...

Embodiment 2

[0028] Genetic testing of Enterococcus faecalis xltyr001.

[0029] Sources of Primers and Primers

[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.

Embodiment 3

[0036] Preparation method of high-concentration tyramide culture solution Pick colonies from the inclined plane and inoculate them into test tubes containing medium A, culture them at 37°C for 24 hours, and the inoculum size in the test tubes 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 to obtain a high-concentration tyramine solution, and the tyramine content of the high-concentration tyramine solution is 4023.94 μg / ml.

[0037] Substratum: MRS liquid medium (with embodiment 1); Described A medium: the MRS liquid medium of 0.1% tyrosine; Described B medium: 0.005% pyridoxal phosphate+0.1% tyrosine acidic MRS broth.

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Abstract

The invention relates to an enterococcus faecalis with the collection number of CGMCC No.3164. 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, thallus is ovate, extends along chain direction, is in pair or in short chain, is Gram-negative, generates lactic acid when fermenting glucose and is elliptic 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, the 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 cultivated 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 of 0.1% of tyrosine, and the culture medium B is an MRS liquid culture medium of 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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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P13/00C12R1/01
Inventor 周光宏卢士玲徐幸莲刘登勇张秋勤
Owner NANJING AGRICULTURAL UNIVERSITY
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