CN101701202A

CN101701202A - Enterococcus faecalis and application thereof

Info

Publication number: CN101701202A
Application number: CN200910232712A
Authority: CN
Inventors: 徐幸莲; 卢士玲; 周光宏; 刘登勇; 韩衍青
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2009-11-27
Filing date: 2009-11-27
Publication date: 2010-05-05
Anticipated expiration: 2029-11-27
Also published as: CN101701202B

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

One Enterococcus faecalis and application thereof

Technical field

The present invention relates to a strain microorganism and an application thereof, belong to biological technical field.

Background technology

The chemical name of tyrasamine is to the hydroxy-beta phenylethylamine, is the important intermediate of synthetic drugs, can be used as biochemical reagents, and at present, the demand of China's tyrasamine and derivative thereof is big, but does not domesticly still have enterprise and can produce qualified product, and is very high from external import price.

Traditional tyrasamine production is to adopt chemical synthesis process, and big for environment pollution, production efficiency is low, is difficult to purify.With tyrosine is precursor, utilizes the tyrosine deearboxylase of microorganisms, produces tyrasamine by microbial metabolism, obtains high density tyrasamine liquid, separates tyrasamine then, can reduce the production cost and the energy consumption of tyrasamine, also becomes the major subjects of research.

Summary of the invention

The objective of the invention is to: at the problem that present tyrasamine production exists, a kind of microorganism is provided and produces tyrasamine, obtain high density tyrasamine liquid by this microbial metabolism.

The object of the present invention is achieved like this: an Enterococcus faecalis (Enterococcus faecium), it is characterized in that: preserving number is CGMCC No.3165, and bacterium colony is cultivated 24h for 37 ℃ on the MRS nutrient agar, enrichment in the MRS liquid nutrient medium, the oyster white bacterium colony, ellipse becomes short chain, Gram-positive, glucose fermentation produces acid, and this bacterial strain is ovalize under simple microscope, and the Genbank number of landing of its 16SrDNA is GQ890354.

In the present invention: described MRS nutrient agar is: described MRS liquid nutrient medium is: peptone 10g, extractum carnis 10g, yeast extract paste 5g, hydrogen citrate three ammonium 2g, glucose 20g, tween 80 1mL, sodium acetate 5g, dipotassium hydrogen phosphate 2g, sal epsom 0.5g, manganous sulfate 0.25g, distilled water 1000mL, pH value 6.2～6.4; Described MRS nutrient agar is to add 1.8% agar at the MRS liquid nutrient medium.

A kind of preserving number is the application of the bacterial strain of CGMCC No.3165, it is characterized in that: be inoculated in the test tube that the A substratum is housed from the inclined-plane picking colony, 37 ℃ leave standstill cultivation 24 hours, and the inoculum size in the test tube is 10 ⁵Cfu/ml.After same medium activates five times repeatedly; Bacterium liquid after the activation at last is inoculated in the B substratum 37 ℃ of static cultivations 4 days, and inoculum size is 10 ⁶Cfu/ml; Bacterium liquid 10000 after cultivating was left the heart 10 minutes, get supernatant liquor, obtain high density tyrasamine liquid, the tyramine content of high density tyrasamine liquid is 4023.94 μ g/ml.

Described A substratum is: the MRS liquid nutrient medium of 0.1% tyrosine; Described B substratum is: the MRS liquid nutrient medium of 0.005% pyridoxal phosphate+0.1% tyrosine.

The invention has the advantages that: because faecium (Enterococcus faecium) xltyr002 product tyrasamine ability is strong, in nutrient solution, tyramine content can reach 4023.94 μ g/ml, and producing tyrasamine with this method will greatly reduce production costs and save energy.

Description of drawings

Fig. 1 is the photo of faecium under simple microscope;

Fig. 2 is the electrophorogram that detects faecium tyrosine deearboxylase gene amplification dna segment;

Fig. 3 is the high-efficient liquid phase chromatogram of biogenic amine standard specimen;

Fig. 4 is the high-efficient liquid phase chromatogram of faecium nutrient solution.

Fig. 5 is the canonical plotting that high performance liquid phase detects tyrasamine.

Embodiment

Embodiment 1

Screening and the preservation of faecium (Enterococcus faecium) xltyr002

Substratum:

The MRS liquid nutrient medium is: peptone 10g, extractum carnis 10g, yeast extract paste 5g, diammonium hydrogen citrate 2g, glucose 20g, tween 80 1mL, sodium acetate 5g, dipotassium hydrogen phosphate 2g, sal epsom 0.5g, manganous sulfate 0.25g, distilled water 1000mL, 6.2～6.4,115 ℃ of sterilizations of pH value 15min.

Lower floor's substratum is: peptone 5g, yeast extract paste 5g, extractum carnis 5g, sodium-chlor 2.5g, glucose 0.5g, tween 1g, sal epsom 0.4g, manganous sulfate 0.03g, dipotassium hydrogen phosphate 2g, Triammonium citrate 2g, lime carbonate 0.1g, ferrous sulfate 0.04g, vitamins B ₁0.01g, pyridoxal phosphate 0.05g, agar 18 grams, tyrosinase 15 gram, 1000ml distilled water.PH5.2,115 ℃ of autoclavings 15 minutes.

The upper strata substratum is: purpurum bromocresolis 0.06g, agar 20g, 1000ml distilled water, pH5.2,121 ℃ of sterilization 10min.

MRS solid medium: in the MRS liquid nutrient medium, add 1.8% agar.

Separation method:

Under aseptic condition, get 20 gram traditional Chinese sausages, shred to be placed on and 180ml is housed in the triangular flask of sterile saline, 230 commentaries on classics shaking tables shook 10 minutes under the room temperature, getting 1ml is inoculated in the MRS liquid nutrient medium, cultivated 24 hours for 37 ℃, get the 1ml nutrient solution and use 9ml physiological saline stepwise dilution, coat on lower floor's substratum, cultivated 72 hours for 37 ℃ to 10-100cfu/ml concentration.Slowly impouring skim upper strata substratum write down the result in 10 minutes, and the bacterium colony that shows purple is positive, and displaing yellow is negative, and is wherein, positive in producing the tyrasamine bacterial strain, negative for not producing the amine bacterial strain.

The picking positive strain is cultivated 24h for 37 ℃ on the MRS solid medium, purifying is three times at least, is inoculated in the MRS liquid nutrient medium and cultivates 24 hours for 37 ℃, and centrifugal back obtains bacterial strain, adds aseptic glycerine, and-80 ℃ of refrigerators are preserved.

This bacterium oyster white bacterium colony, ellipse becomes short chain, Gram-positive, glucose fermentation produces acid, and this bacterial strain is oval under simple microscope.

This bacterial strain is according to Gen bank comparison result, called after faecium (Enterococcusfaecium) xltyr002, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 6th, 2009, culture presevation number is CGMCC No.3165, and the Genbank number of landing of its 16S rDNA is GQ890354.

Embodiment 2

The gene test of faecium (Enterococcus faecium) xltyr002.

Primer

TD2：5‘-ACATAGTCAACCATRTTGAA-3’；

TD5：5‘-CAAATGGAAGAAGAAGTAGG-3’；

Detection method:

25 μ l reaction systems comprise: GoTaq Green Master Mix 12.5 μ l, every kind of each 1.0 μ l of primer, DNA masterplate 2 μ l, deionization distilled water 8.5 μ l.

The pcr amplification program: 94 ℃ of pre-sex change 5min, 35 circulations comprise: 94 ℃ of sex change 45s, 48 ℃ of annealing 45s, 72 ℃ are extended 60s, and final 72 ℃ are extended 7min, are cooled to 4 ℃.

With the Auele Specific Primer TD2 and the TD5 of the tyrosine deearboxylase gene that can increase, can successfully amplify the gene fragment of 1100bp, in Fig. 2, as seen the gene segment that swimming lane 1 obtains for the faecium DNA cloning, exists the tyrosine deearboxylase gene in the faecium.

Embodiment 3

High density tyrasamine inoculum preparation method is inoculated in the test tube that the A substratum is housed from the inclined-plane picking colony, and 37 ℃ leave standstill cultivation 24 hours, and the inoculum size in the test tube is 10 ⁵Cfu/ml.Activate five faeciums repeatedly in same medium and be inoculated in the B substratum 37 ℃ of static cultivations 4 days, inoculum size is 10 ⁶Cfu/ml; Bacterium liquid 10000 after cultivating was left the heart 10 minutes, get supernatant liquor, obtain high density tyrasamine liquid, the tyramine content of high density tyrasamine liquid is 4023.94 μ g/ml.

Substratum: MRS liquid nutrient medium (with embodiment 1); Described A substratum is: the MRS liquid nutrient medium that adds 0.1% tyrosine; Described B substratum is: the MRS liquid nutrient medium that adds 0.005% pyridoxal phosphate+0.1% tyrosine.

Embodiment 4.

Tyramine content detection method (high performance liquid phase detection)

1, the preparation of biogenic amine standardized solution

Accurately take by weighing each 50mg of tryptamines, phenylethylamine, tyrasamine, cadaverine, putrescine, spermidine and spermine, with the perchloric acid (HClO of 0.4mol/L ₄) be settled to 50mL, it is standby to make the 1mg/ml storing solution.Get above standard substance storing solution respectively, use 0.4mol/L HClO ₄Be mixed with final concentration and be respectively 5.0,10,20,30,40,50 μ g/ml standardized solution, lucifuge, 4 ℃ of refrigerators are preserved.

2, the derivatize of sample solution

The sample 1mL that gets embodiment 3 is in the 5ml volumetric flask, the 2N NaOH that adds 200 μ L makes it to be alkalescence, the saturated sodium bicarbonate solution that adds 300 μ L then cushions, (concentration is 10mg/mL to add dansyl chloride (dansyl chloride) solution of 2ml again, solvent is an acetone), be positioned over reaction treatment 40min in 40 ℃ of water-bath dark then.Reaction finishes the ammoniacal liquor stopped reaction that the back adds 100 μ L, removes residual dansyl chloride solution.Use the acetonitrile constant volume to 5mL at last.Behind organic membrane filtration with 0.22 μ m after the derivation process, obtain the derivative of sample solution.

3, the derivatize of standardized solution standard specimen

Get 5.0,10,20,30,40,50 μ g/ml standardized solution sample 1mL respectively in the 5ml volumetric flask, the 2N NaOH that adds 200 μ L makes it to be alkalescence, the saturated sodium bicarbonate solution that adds 300 μ L then cushions, (concentration is 10mg/mL to add dansyl chloride (dansyl chloride) solution of 2ml again, solvent is an acetone), be positioned over reaction treatment 40min in 40 ℃ of water-bath dark then.Reaction finishes the ammoniacal liquor stopped reaction that the back adds 100 μ L, removes residual dansyl chloride solution.Use the acetonitrile constant volume to 5mL at last.Behind organic membrane filtration with 0.22 μ m after the derivation process, obtain the derivative of six groups of sample solutions.

4, detection method is:

Waters Alliance2695 Liquid Detection system, chromatographic column is AgilentZORBAXXDB-C18 (4.6 * 250mm2,5 μ m), flow velocity is 1mLmin-1, and the ultraviolet detection wavelength is 254nm, sample size 20 μ L, 30 ℃ of column temperatures, mobile phase A are water, and Mobile phase B is an acetonitrile, adopt gradient elution, elution program sees Table 1.

Table 1 gradient elution program

In the high performance liquid phase figure of the biogenic amine standard specimen of Fig. 3: 1 is tryptamines, and 2 is phenylethylamine, and 3 is putrescine, and 4 is cadaverine, and 5 is histamine, and 6 is tyrasamine, and 7 is spermidine, and 8 is spermine.Sample solution high performance liquid phase detected result of the present invention as shown in Figure 4.As seen from Figure 4, contain tyrasamine in the sample solution.

Utilize above-mentioned detection method to detect, the value of meeting with a response is 9408270.896 (referring to Fig. 5), calculating the result according to typical curve is 402.394 μ g/mg, but owing in testing process, former state has been diluted 10 times, so final tyramine content is 4023.94ug/mg.

More than each embodiment be not to concrete qualification of the present invention.

Claims

1. an Enterococcus faecalis (Enterococcus faecium), it is characterized in that: preserving number is CGMCC No.3165, and bacterium colony is cultivated 24h for 37 ℃ on the MRS nutrient agar, enrichment in the MRS liquid nutrient medium, the oyster white bacterium colony, ellipse becomes short chain, Gram-positive, glucose fermentation produces acid, and this bacterial strain is ovalize under simple microscope, and the Genbank number of landing of its 16S rDNA is GQ890354.

2. an Enterococcus faecalis according to claim 1 (Enterococcus faecium) is characterized in that: described MRS liquid nutrient medium is: peptone 10g, extractum carnis 10g, yeast extract paste 5g, hydrogen citrate three ammonium 2g, glucose 20g, tween 80 1mL, sodium acetate 5g, dipotassium hydrogen phosphate 2g, sal epsom 0.5g, manganous sulfate 0.25g, distilled water 1000mL, pH value 6.2～6.4; Described MRS nutrient agar is to add 1.8% agar at the MRS liquid nutrient medium.

3. application that preserving number is the bacterial strain of CGMCC No.3165 is characterized in that: be inoculated in the test tube that the A substratum is housed from the inclined-plane picking colony, 37 ℃ leave standstill cultivation 24 hours, and the inoculum size in the test tube is 10 ⁵Cfu/ml.After same medium activates five times repeatedly; Bacterium liquid after the activation at last is inoculated in the B substratum 37 ℃ of static cultivations 4 days, and inoculum size is 10 ⁶Cfu/ml; Bacterium liquid 10000 after cultivating was left the heart 10 minutes, get supernatant liquor, obtain high density tyrasamine liquid, the tyramine content of high density tyrasamine liquid is 4023.94 μ g/ml.

4. the application that utilizes preserving number for the bacterial strain of CGMCC No.3165 according to claim 3 is characterized in that: described A substratum is: the MRS liquid nutrient medium that adds 0.1% tyrosine; Described B substratum is: the MRS liquid nutrient medium that adds 0.005% pyridoxal phosphate+0.1% tyrosine.