CN103060232B - Enterococcus faecalis synthesizing osmophilic ethyl carbamate hydrolase and applications thereof - Google Patents
Enterococcus faecalis synthesizing osmophilic ethyl carbamate hydrolase and applications thereof Download PDFInfo
- Publication number
- CN103060232B CN103060232B CN201210578408.8A CN201210578408A CN103060232B CN 103060232 B CN103060232 B CN 103060232B CN 201210578408 A CN201210578408 A CN 201210578408A CN 103060232 B CN103060232 B CN 103060232B
- Authority
- CN
- China
- Prior art keywords
- enterococcus faecalis
- enzyme
- ethyl carbamate
- urethane ester
- ester hydrolase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Enzymes And Modification Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention discloses an Enterococcus faecalis SW08 for synthesizing acid ethyl carbamate hydrolase, which has been preserved in China Typical Culture Collection Center on October 19, 2012, and the accession number is CCTCC No.M2012417. The Enterococcus faecalis SW08 can rapidly degrade ethyl carbamate and urea in an acidic environment, which plays a positive role in elimination of ethyl carbamate in fermented foods.
Description
Technical field
The present invention relates to a kind of enterococcus faecalis and application thereof, enterococcus faecalis and application thereof that particularly the synthetic resistance to height of a kind of energy oozes urethane ester hydrolase.
Background technology
Urethanum (Ethyl Carbamate, EC), is medically having important use, is once once being used as treating multiple myeloma and chronic lymphocytic leukemia, and is using as narcotic.Research in recent years discovery, urethanum is a kind of mankind's potential carcinogen matter, can cause the diseases such as lung cancer, lymphatic cancer, liver cancer, international cancer research institution classified urethanum as 2A class carcinogens in 2007 as.Urethanum is the by product producing in the production process of a kind of concomitant fermentations food (as alcoholic beverage, beans sauce, soy sauce etc.), and food safety and organism health are all had a serious impact and endangered.The food-safety problem causing in the face of urethanum, with microorganism and enzyme liberating realize urethanum in food without harmization, be comparatively ideal solution.
Summary of the invention
Technical problem to be solved by this invention is to provide enterococcus faecalis (Enterococcus faecals) SW08 that the synthetic resistance to height of a strain oozes urethane ester hydrolase, on October 19th, 2012, be preserved in Chinese Typical Representative microbial strains preservation administrative center, deposit number is CCTCC M2012417, and preservation address is Wuhan, China Wuhan University.Bacterium colony is that white is circular, and surface drying is smooth, and regular edges is opaque, bacterium colony size 1-2mm, amphimicrobian or anaerobism, available nutrient broth medium aerated culture or with the standing cultivation of MRS substratum anaerobism.Thalline Gram-positive, has pod membrane, and under 1000 * microscope, observation of cell is spherical in shape.
Described enterococcus faecalis is separated from the female mice gi tract of growing up, its storage conditions is as follows: from well-grown flat board, picking list bacterium colony is transferred in nutrient broth medium, at 30 ℃, 200rpm, cultivate 16h, get 500 μ L nutrient solutions and move in the preservation pipe that contains 500 μ L40% glycerine, be placed in-80 ℃ of Refrigerator stores.
Above-mentioned nutrient broth medium is two type nutrient broth mediums, fills a prescription as peptone 10g, and extractum carnis 10g, sodium-chlor 5g, distilled water 1L, as need solid medium, then add 2% agar.
Another object of the present invention is to provide the extracting method of above-mentioned enterococcus faecalis crude enzyme liquid.
Another object of the present invention is to provide the application of above-mentioned enterococcus faecalis, and its synthetic resistance to height oozes urethane ester hydrolase and can be greater than under the condition under 15% condition the by product urethanum in fast decoupled leavened food and produce ethanol and ammonia in NaCl concentration.It has the highest enzyme 11U/g that lives when optimal pH 7.0, is greater than under 15% condition in NaCl concentration, and the resistance to height that fusiform Methionin genus bacillus produces oozes urethane ester hydrolase and can keep 40% enzyme to live.
Enterococcus faecalis provided by the invention can synthesize resistance to height and ooze urethane ester hydrolase, at pH, is to have the highest enzyme at 7.0 o'clock to live, and can ooze fast decoupled urethanum under condition at height.Under the condition that is 7.0 at pH, its urethane ester hydrolase enzyme is lived as 11U/g, is greater than under 15% condition in NaCl concentration, and it can keep 40% urethane ester hydrolase enzyme to live.
Accompanying drawing explanation
Fig. 1 enterococcus faecalis Photomicrograph
The impact that Fig. 2 pH lives on enterococcus faecalis urethane ester hydrolase enzyme
The tolerance of Fig. 3 enterococcus faecalis urethane ester hydrolase to NaCl
Embodiment
Embodiment 1: enterococcus faecalis screening method
The urethane ester solution (200mg/kg body weight/day) of raising by force that every 5 weeks large female kunming mice of 25g is carried out five days is dissected afterwards, and the normal saline flushing mouse intestinal tissue that is 0.9% by concentration, get suspensions of tissues 1mL centrifugal 10min under 1000 * g, get supernatant liquor and be inoculated in (beef extract 10g, peptone 10g, NaCl5g in enrichment medium, urethanum 10g, water 1000mL, pH7.0), aerated culture 20h at 30 ℃.Nutrient solution, in the centrifugal 5min of 2000 * g, is got supernatant and is coated the middle cultivation of screening culture medium (beef extract 10g, peptone 10g, NaCl5g, urethanum 30g, agar 20g, water 1000mL, pH5.0) three days.The bacterial strain that choosing colony form is larger carries out preservation after transferring to and cultivating 20h in nutrient broth medium.
The bacterial strain of picking-80 ℃ preservation is rule on nutrient broth agar solid medium, in 37 ℃, cultivate after 24h, picking list colony inoculation is in nutrient broth liquid nutrient medium, 37 ℃ are 200rpm at rotating speed shaking table is cultivated 24h, centrifugal 5min (12000rpm, 4 ℃) rear collection thalline, then use the phosphate buffered saline buffer resuspension thalline of pH7.0 to 0.1g/mL.By ultrasonication, carry out the extraction of enzyme extract, crude enzyme liquid is carried out to urethane ester hydrolase enzyme activity determination and the bacterial strain that a strain has urethanum hydrolytic enzyme activities detected, by known this bacterial strain of 16S rDNA sequential analysis, it is a strain enterococcus faecalis, on October 19th, 2012, be preserved in Chinese Typical Representative microbial strains preservation administrative center, deposit number is CCTCC M2012417, and preservation address is Wuhan, China Wuhan University.
Embodiment 2: the extracting method of urethane ester hydrolase crude enzyme liquid in enterococcus faecalis
The bacterial strain of picking-80 ℃ preservation is rule on nutrient broth agar solid medium, in 37 ℃, cultivate after 24h, picking list colony inoculation is in nutrient broth liquid nutrient medium, 37 ℃ are 200rpm at rotating speed shaking table is cultivated 24h, collect bacterium liquid 50mL low-temperature centrifugation 10min (10000rpm in high-speed refrigerated centrifuge, 4 ℃) after collect thalline, in bacterial sediment, add the long-pending 50mM PBS of identical bacteria liquid or 50mM Tris-HCl(pH of buffer to choose according to screened protein stabilized scope) resuspended washing three times.Use again the PBS damping fluid suspension thalline of pH7.0 to 0.1g/mL.By bacterium liquid-80 ℃ freezing, room temperature is melted, multigelation three times, because ice pellets in cell forms and the salt concn of remaining cell liquid increases and causes swelling, makes cellularstructure fragmentation.By the bacterium liquid of multigelation, be placed in ice-water bath and carry out ultrasonication (can add appropriate N,O-Diacetylmuramidase if desired), ultrasound condition: 25W, work 2S, interval 2S, repeated work 30min, until the change of thalline solution is limpid, about spended time 60min.By after thalline ultrasonication in refrigerated centrifuge centrifugal 10min (10000rpm, 4 ℃), supernatant liquor is transferred in clean Eppendorf pipe to cryopreservation.
Embodiment 3: the enzyme detection method alive of urethane ester hydrolase crude enzyme liquid
The principle of urethane ester hydrolase crude enzyme liquid enzyme detection method alive is to utilize under certain condition, and urethanum degrading enzyme section decomposes urethanum to generate ethanol, ammonia and carbonic acid gas.It is aobvious blue that ammonia and phenol-sodium hypochlorite reaction form indophenol blue, surveys its light absorption value with spectrophotometer at 630nm place, analyzes the growing amount of ammonia, thereby it is alive to obtain the enzyme of urethane ester hydrolase.
The preparation of enzyme activity determination reagent:
Developer I: take 15g phenol and 0.625g sodium nitroprusside is settled to 250mL with ultrapure water
Developer II: take 13.125g sodium hydroxide and 7.5mL clorox is settled to 250mL with ultrapure water
Terminator: take 10g trichoroacetic acid(TCA) and be settled to 100mL with ultrapure water
NH
4 +standardized solution preparation: 1mL ammoniacal liquor is dissolved in ultrapure water and is settled to 145mL, is made into the NH of 0.1mol/L
4 +solution, and as mother liquor, with ultrapure water by it dilution and be mixed with the NH of 0.1mmol/L, 0.2mmol/L, 0.3mmol/L, 0.4mmol/L, 0.5mmol/L
4 +standardized solution.
The drafting of ammonium ion typical curve: accurately pipette respectively 1mL NH
4 +normal gradients liquid is placed in the 10mL colorimetric cylinder of serial number.At 37 ℃, constant temperature insulation 15min, draws 1mL terminator immediately in colorimetric cylinder, and vibration mixes.Add successively 1mL developer I and developer II, vibration, makes it fully to mix strongly again, reaction 20min.Ultrapure water is settled to 10mL, and colorimetric estimation OD value under 630nm, take OD value as ordinate zou, NH
4 +gradient is X-coordinate mapping, obtains typical curve.
The mensuration that enzyme is lived
Get two 10mL colorimetric cylinders, add respectively 1mL enzyme liquid and ultrapure water.Then in two pipes, add respectively 1mL ultrapure water, in 37 ℃ of constant water bath box, react after 15min, at two pipes, respectively add 1mL trichoroacetic acid(TCA) terminator, the developer I and the 1mL developer II that after mixing, add 1mL, strong concussion, takes out after continuation is incubated 20min in 37 ℃ of constant water bath box, with ultrapure water, is diluted to 10mL, 630nm place colorimetric, and record OD value.
Enzyme calculation formula alive: enzyme activity=Δ OD
630* n * k * 10/15
In formula: Δ OD
630: after enzyme reaction, sample determination and blank test optical density value is poor
N: enzyme activity determination liquid extension rate
K: the inverse of slope of standard curve
10:1mL sample liquid is diluted to the multiple of 10mL
15: the time of enzyme reaction (min) (time of enzyme reaction is 15min)
Enzyme is lived and is defined
Enzyme activity definition: per minute bottom exploded deposits yields 1 μ mol ammonia is an enzyme activity unit under normal pressure, 37 ℃ of conditions.
Embodiment 4: the determining of urethane ester hydrolase crude enzyme liquid optimal pH
According to the form below configuration buffered soln, its pH is as the criterion with acidometer measured value.
Prepare two groups of each 7 10mL colorimetric cylinders, in first group of 7 colorimetric cylinder, corresponding damping fluid in every all adds on 8mL table, then add 3% urethane ester solution 1mL, add a certain amount of urethane ester hydrolase crude extract (extension rate of enzyme and add-on will be selected suitably, so as under experiment condition at that time, to obtain in linearity range absorbance value A
630.7 test tubes of another group are also every and all add corresponding damping fluid in the upper table of 8mL, but no longer add urethanum solution and add the deionized water of equivalent, respectively the control tube when measuring.All test tubes all water are supplied 10mL.By enzyme activity determination method in above-described embodiment three, carrying out reaction detection enzyme lives.
The enzyme of take (U/g) alive is ordinate zou, take pH as X-coordinate, draws the relation curve of lower urethanum hydrolytic enzyme activities and pH.Optimal pH or the pH scope of analytic curve definite urethane ester hydrolase.Accompanying drawing 2 is shown in by the collection of illustrative plates that drafting obtains.
Embodiment five: the research of urethanum crude enzyme liquid salt tolerance
Enzyme liquid is respectively to 0% in NaCl concentration, 5%, 10%, 15%, in 18% damping fluid, under enzyme is lived stable lesser temps, be incubated 20min, the enzyme of measuring under corresponding conditions according to the method in embodiment tri-is lived, draw the relation curve of salt concn-remnant enzyme activity and see accompanying drawing 3, determine the salt stability of enzyme.
Claims (3)
- The synthetic resistance to height of one strain ooze urethane ester hydrolase enterococcus faecalis SW08 ( enterococcus faecalissW08), on October 19th, 2012, be preserved in Chinese Typical Representative culture collection center, deposit number is CCTCC NO:M2012417.
- 2. enterococcus faecalis claimed in claim 1 is applied to the production of urethane ester hydrolase.
- 3. enterococcus faecalis claimed in claim 1 is applied to the degraded of urethanum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210578408.8A CN103060232B (en) | 2012-12-27 | 2012-12-27 | Enterococcus faecalis synthesizing osmophilic ethyl carbamate hydrolase and applications thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210578408.8A CN103060232B (en) | 2012-12-27 | 2012-12-27 | Enterococcus faecalis synthesizing osmophilic ethyl carbamate hydrolase and applications thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103060232A CN103060232A (en) | 2013-04-24 |
| CN103060232B true CN103060232B (en) | 2014-04-09 |
Family
ID=48103132
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210578408.8A Active CN103060232B (en) | 2012-12-27 | 2012-12-27 | Enterococcus faecalis synthesizing osmophilic ethyl carbamate hydrolase and applications thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103060232B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105929179B (en) * | 2016-06-01 | 2017-12-12 | 中国农业科学院兰州兽医研究所 | A kind of enterococcus faecalis indirect hemagglutination detection kit and its application |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6620585B1 (en) * | 2000-08-02 | 2003-09-16 | Elitra Pharmaceuticals, Inc. | Use of ectoenzymes and secreted enzymes to monitor cellular proliferation |
| WO2005047514A1 (en) * | 2003-11-10 | 2005-05-26 | University Of Kent | Proteins involved in signal transduction |
| WO2010020657A1 (en) * | 2008-08-19 | 2010-02-25 | Profos Ag | Artificial peptidoglycan lysing enzymes and peptidoglycan binding proteins |
| CN102511663A (en) * | 2012-01-09 | 2012-06-27 | 信息产业电子第十一设计研究院科技工程股份有限公司 | Straw microorganism starter |
-
2012
- 2012-12-27 CN CN201210578408.8A patent/CN103060232B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6620585B1 (en) * | 2000-08-02 | 2003-09-16 | Elitra Pharmaceuticals, Inc. | Use of ectoenzymes and secreted enzymes to monitor cellular proliferation |
| WO2005047514A1 (en) * | 2003-11-10 | 2005-05-26 | University Of Kent | Proteins involved in signal transduction |
| WO2010020657A1 (en) * | 2008-08-19 | 2010-02-25 | Profos Ag | Artificial peptidoglycan lysing enzymes and peptidoglycan binding proteins |
| CN102511663A (en) * | 2012-01-09 | 2012-06-27 | 信息产业电子第十一设计研究院科技工程股份有限公司 | Straw microorganism starter |
Non-Patent Citations (4)
| Title |
|---|
| 两株降胆固醇球菌生长条件的研究;李春 等;《现代食品科技》;20051230;第21卷(第04期);28-30 * |
| 刘志远 等.肠球菌氨基糖苷类高水平耐药基因的检测.《中华检验医学杂志》.2005,第28卷(第01期),96-99. |
| 李春 等.两株降胆固醇球菌生长条件的研究.《现代食品科技》.2005,第21卷(第04期),28-30. |
| 肠球菌氨基糖苷类高水平耐药基因的检测;刘志远 等;《中华检验医学杂志》;20050130;第28卷(第01期);96-99 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103060232A (en) | 2013-04-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103074275B (en) | Lysinibacillus fusiformis for producing ethyl urethane hydrolase and application of lysinibacillus fusiformis | |
| CN103114064B (en) | Marine actinomycete with antibacterial activity to multiple plant pathogens | |
| CN102161975B (en) | Streptomyces sp. GSDX-1318, and fermentation method for producing oligosaccharide antibiotic avilamycin | |
| CN101597578B (en) | Enramycin producing strain and extraction method by using macroporous resin | |
| CN101870740B (en) | Morchellaconica extracellular polysaccharide extractive and preparation method and application thereof | |
| CN100368530C (en) | Bifidobacteria exocellular polysaccharide and its production method and special purpose production strain | |
| CN104087628A (en) | Method for reducing viscosity of gamma-polyglutamic acid fermentation liquid | |
| CN103667097B (en) | Bacillus sp.N11-8 and active component thereof having antitumor action | |
| CN102994433B (en) | Acid producing klebsiella oxytoca for synthesizing acidity ethyl carbamate hydrolase and application thereof | |
| CN113265337B (en) | Marine aspergillus versicolor and isolated culture method and application thereof | |
| CN103045514B (en) | Proteus mirabilis for synthetizing acidic ethyl urethane hydrolytic enzyme and application thereof | |
| CN100415870C (en) | Nie's brevibacterium new strain and process for preparing enzyme by it | |
| CN103074234A (en) | Marine fungus aspergillus sydowii and application thereof to preparation of anti-tumor medicines | |
| CN103060232B (en) | Enterococcus faecalis synthesizing osmophilic ethyl carbamate hydrolase and applications thereof | |
| CN102787089B (en) | Acinetobacterbeijerinckii and application of acinetobacterbeijerinckii | |
| CN102229905B (en) | Streptomyces ahygroscopic Str-8 and method for preparing epsilon-polylysine and salts thereof by using same | |
| CN103865850A (en) | Vampirovibrio sp. and method for preparing agarase | |
| CN104046584A (en) | Bifidobacterium adolescentis bacteriocin as well as production method and special production strain of bifidobacterium adolescentis bacteriocin | |
| CN104745554B (en) | Bacillus produces the fermentation medium and fermentation process of protease and gemma | |
| CN104087523A (en) | Marine Streptomyces, method for preparation of Enterocin therefrom and application of the same | |
| CN103013948B (en) | Method for producing ethyl carbamate enzyme by purifying lysinibacillus fusiformis | |
| CN102994484A (en) | Culture medium and method for fermentation production of chitinase by aschersonia placenta | |
| CN105802874A (en) | Mixed bacterium for efficiently degrading atrazine and fermental culture method | |
| CN103642736B (en) | A kind of bacterial strain and screening method thereof and application | |
| CN102409012A (en) | Klebsiella pneumonia being separated from Termite gut |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CB03 | Change of inventor or designer information |
Inventor after: Chen Jian Inventor after: Du Guocheng Inventor after: Zhang Jiran Inventor after: Fang Fang Inventor before: Chen Jian Inventor before: Du Guocheng Inventor before: Fang Fang Inventor before: Zhang Jiran |
|
| COR | Change of bibliographic data | ||
| CP02 | Change in the address of a patent holder | ||
| CP02 | Change in the address of a patent holder |
Address after: 214122 Jiangsu city of Wuxi Province District Liangxi No. 898 South Road 7 layer beam Creek area of food science and Technology Park Patentee after: Jiangnan University Address before: 1800 No. 214122 Jiangsu city of Wuxi Province Li Lake Avenue Patentee before: Jiangnan University |