CN103074275A - Lysinibacillus fusiformis for producing ethyl urethane hydrolase and application of lysinibacillus fusiformis - Google Patents
Lysinibacillus fusiformis for producing ethyl urethane hydrolase and application of lysinibacillus fusiformis Download PDFInfo
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- CN103074275A CN103074275A CN2012105784247A CN201210578424A CN103074275A CN 103074275 A CN103074275 A CN 103074275A CN 2012105784247 A CN2012105784247 A CN 2012105784247A CN 201210578424 A CN201210578424 A CN 201210578424A CN 103074275 A CN103074275 A CN 103074275A
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Abstract
The invention discloses lysinibacillus fusiformis SC02 for producing ethyl urethane hydrolase. The lysinibacillus fusiformis SC02 was collected into China Center for Type CultureCollection (CCTCC) on October 19th, 2012, wherein the collection number is CCTCC NO: M2012414. The lysinibacillus fusiformis SC02 can quickly degrade ethyl urethane under the hypertonic environment (the NaCl concentration is more than 15 percent), achieves active effect of eliminating the ethyl urethane in fermented food and has good application prospect.
Description
Technical field
The present invention relates to fusiformis Methionin genus bacillus and application thereof that the synthetic anti-height of a kind of fusiformis Methionin genus bacillus and application thereof, particularly a kind of energy oozes the urethane ester hydrolase.
Background technology
Urethanum (Ethyl Carbamate, EC) is medically having important use, once once is being used as treating multiple myeloma and chronic lymphocytic leukemia, and is using as narcotic.Research in recent years finds that urethanum is a kind of human potential carcinogen matter, can cause the diseases such as lung cancer, lymphatic cancer, liver cancer, and international cancer research institution classified urethanum as 2A class carcinogens in 2007.Urethanum is the by product that produces in the production process of a kind of concomitant fermentations food (such as alcoholic beverage, beans sauce, soy sauce etc.), and food safety and organism health are seriously influenced and endangers.In the face of the food-safety problem that urethanum causes, with the nothing harmization of urethanum in microorganism and the enzyme liberating realization food, be comparatively ideal solution.
Summary of the invention
Technical problem to be solved by this invention provides fusiformis Methionin genus bacillus (Lysinibacillus fusiformis) SC02 that the urethane ester hydrolase is produced in a strain, be preserved in Chinese allusion quotation shape microbial strains preservation administrative center on October 19th, 2012, deposit number is CCTCC NO:M2012414.Bacterium colony is that the wax sample is opaque, and the edge is irregular, colony diameter 2-4mm, and cultivating 1-2 days bacterium colonies is white, colony colour is deepened gradually after 3 days, finally aobvious burgundy.Aerobic or amphimicrobian.The thalline Gram-positive, without pod membrane, 1000 * microscopically observation of cell is shaft-like fusiformis, and there is oval gemma on part thalline top.
Described fusiformis Methionin genus bacillus separates from the female mice enteron aisle of growing up, its storage conditions is as follows: picking list bacterium colony is transferred the nutrition broth culture from well-grown flat board, at 30 ℃, 200rpm, cultivate 16h, get the immigration of 500 μ L nutrient solutions and contain in the preservation pipe of 500 μ L40% glycerine, place-80 ℃ of Refrigerator stores.
Above-mentioned nutrient broth medium is the dimorphism nutrient broth medium, fills a prescription to be peptone 10g, and extractum carnis 10g, sodium-chlor 5g, distilled water 1L such as the need solid medium, adds 2% agar again.
Another object of the present invention provides the application of above-mentioned fusiformis Methionin genus bacillus, its synthetic anti-height ooze the urethane ester hydrolase can NaCl concentration greater than the condition under 15% the condition under in the fast decoupled leavened food the by product urethanum and produce ethanol and ammonia.It has the highest enzyme 40U/g that lives when optimal pH 8.0, NaCl concentration greater than 15% condition under, the anti-height that fusiformis Methionin genus bacillus produces oozes the urethane ester hydrolase and can keep 20% enzyme to live.
Fusiformis Methionin genus bacillus provided by the invention can be synthesized anti-height and be oozed the urethane ester hydrolase, is to have the highest enzyme at 8.0 o'clock to live at pH, can ooze fast decoupled urethanum under the condition at height.Be that its urethane ester hydrolase enzyme is lived and is 40U/g under 8.0 the condition at pH, NaCl concentration greater than 15% condition under, it can keep 20% urethane ester hydrolase enzyme to live.
Figure of description
Fig. 1 fusiformis Methionin genus bacillus Photomicrograph
The impact that Fig. 2 pH lives on fusiformis Methionin genus bacillus urethane ester hydrolase enzyme
Fig. 3 fusiformis Methionin genus bacillus urethane ester hydrolase is to the tolerance of NaCl
Embodiment
Embodiment 1: fusiformis Methionin genus bacillus screening method
The urethane ester solution (200mg/kg body weight/day) of raising by force that every 5 all large female kunming mice of 25g carried out five days is dissected afterwards, and be 0.9% normal saline flushing mouse intestinal tissue with concentration, get suspensions of tissues 1mL under 1000 * g centrifugal 10 minutes, get supernatant liquor and be inoculated in (beef extract 10g, peptone 10g, NaCl5g in the enrichment medium, urethanum 10g, water 1000mL, pH7.0), 30 ℃ of lower aerated culture 20h.Nutrient solution 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 in the centrifugal 5min of 2000 * g.The bacterial strain that the choosing colony form is larger carries out preservation after transferring to and cultivating 20h in the nutrient broth medium.
The bacterial strain of picking-80 ℃ preservation is rule at the nutrient broth agar solid medium, behind 37 ℃ of cultivation 24h, picking list colony inoculation is in the nutrient broth liquid nutrient medium, 37 ℃ is the shaking table cultivation 24h of 200rpm at rotating speed, centrifugal 5min (12000rpm, 4 ℃) the rear thalline of collecting, use again the phosphate buffered saline buffer resuspension thalline of pH7.0 to 0.1g/mL.Carry out the extraction of enzyme extract by ultrasonication, crude enzyme liquid is carried out urethane ester hydrolase enzyme activity determination detect the bacterial strain that a strain has the urethanum hydrolytic enzyme activities, by 16S rDNA sequential analysis as can be known this bacterial strain be a strain fusiformis Methionin genus bacillus, be preserved in Chinese allusion quotation shape microbial strains preservation administrative center on October 19th, 2012, deposit number is CCTCC NO:M2012414, and the preservation address is Wuhan, China Wuhan University.
Embodiment 2: the extracting method of urethane ester hydrolase crude enzyme liquid
The bacterial strain of picking-80 ℃ preservation is rule at the nutrient broth agar solid medium, behind 37 ℃ of cultivation 24h, picking list colony inoculation is in the nutrient broth liquid nutrient medium, 37 ℃ is the shaking table cultivation 24h of 200rpm at rotating speed, collect bacterium liquid 50mL low-temperature centrifugation 10min (10000rpm in high-speed refrigerated centrifuge, 4 ℃) after collect thalline, adding identical bacteria liquid long-pending 50mM PBS or 50mM Tris-HCl(pH of buffer in the bacterial sediment should choose according to the protein stabilized scope of screening) resuspended washing three times.Use again the phosphate buffered saline buffer suspension thalline of pH7.0 to 0.1g/mL.With bacterium liquid-80 ℃ freezing, room temperature is melted, multigelation three times because ice pellets forms and the salt concn of remaining cell liquid increases and causes swelling in the cell, makes the cellularstructure fragmentation.With the bacterium liquid of multigelation, place ice-water bath to carry out ultrasonication (can suitably add a small amount of N,O-Diacetylmuramidase and accelerate broken speed), ultrasound condition: 25W, work 2S, interval 2S, repeated work 30min, until the thalline solution becomes limpid till, about spended time 60min.With after the thalline ultrasonication in refrigerated centrifuge centrifugal 10min (10000rpm, 4 ℃), supernatant liquor is transferred in the clean Eppendorf pipe cryopreservation.
Embodiment 3: the enzyme biopsy survey method of urethane ester hydrolase crude enzyme liquid
The principle of urethane ester hydrolase crude enzyme liquid enzyme biopsy survey method is to utilize under certain condition, and urethanum degrading enzyme section decomposes generation ethanol, ammonia and carbonic acid gas with urethanum.It is aobvious blue that ammonia and phenol-sodium hypochlorite reaction forms indophenol blue, surveys its light absorption value at the 630nm place with spectrophotometer, analyzes the growing amount of ammonia, thereby the enzyme that obtains the urethane ester hydrolase is lived.
The preparation of enzyme activity determination reagent:
Developer I: take by weighing 15g phenol and the 0.625g sodium nitroprusside is settled to 250mL with ultrapure water
Developer II: take by weighing 13.125g sodium hydroxide and the 7.5mL clorox is settled to 250mL with ultrapure water
Terminator: take by weighing the 10g trichoroacetic acid(TCA) and be settled to 100mL with ultrapure water
NH
4 +The standardized solution preparation: 1mL ammoniacal liquor is dissolved in the 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 with 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 places the 10mL colorimetric cylinder of serial number.37 ℃ of lower constant temperature insulation 15min draw the 1mL terminator in colorimetric cylinder, the vibration mixing immediately.Add successively 1mL developer I and developer II, strong vibration makes it abundant mixing again, reaction 20min.Ultrapure water is settled to 10mL, colorimetric estimation OD value under 630nm, and take the OD value as ordinate zou, NH
4 +Gradient is the 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 the 1mL ultrapure water, after in 37 ℃ of constant water bath box, reacting 15min, respectively add 1mL trichoroacetic acid(TCA) terminator at two pipes, the developer I and the 1mL developer II that add 1mL behind the mixing, strong concussion continues to take out behind the insulation 20min in 37 ℃ of constant water bath box, is diluted to 10mL with ultrapure water, 630nm place colorimetric, and record OD value.
Formula is calculated in enzyme work: enzyme activity=Δ OD
630* n * k * 10/15
In the formula: Δ OD
630: sample determination and blank test optical density value is poor after the enzyme reaction
N: enzyme activity determination liquid extension rate
K: the inverse of slope of standard curve
The 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 the acidometer measured value.
Prepare two groups of each 7 10mL colorimetric cylinders, in first group of 7 colorimetric cylinder, all add corresponding damping fluid in the 8mL table at every, 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 the linearity range absorbance value A
630).7 test tubes of another group also are every and all add corresponding damping fluid in the upper table of 8mL, but no longer add amino ethyl formate solution and add the deionized water of equivalent, respectively the control tube when measuring.All test tubes all water are supplied 10mL.Carrying out the reaction detection enzyme by enzyme activity determination method in above-described embodiment three lives.
Live (U/g) as ordinate zou take enzyme, take pH as X-coordinate, draw the relation curve of lower urethanum hydrolytic enzyme activities and pH.Optimal pH or the pH scope (Fig. 2) of analytic curve and definite urethane ester hydrolase.
Embodiment 5: the research of urethanum crude enzyme liquid salt tolerance
Enzyme liquid is respectively 0%, 5% in NaCl concentration, 10%, in 15%, 18% the damping fluid, under living stable lesser temps, enzyme is incubated 20min, live according to the enzyme that the method among the embodiment three is measured under the corresponding conditions, draw the relation curve of salt concn-remnant enzyme activity and see Fig. 3, determine the salt stability of enzyme.
Claims (5)
1. the fusiformis Methionin genus bacillus SC02 (Lysinibacillus fusiformis SC02) of urethane ester hydrolase is produced in a strain, be preserved in Chinese allusion quotation shape culture collection center on October 19th, 2012, deposit number is CCTCC No:M2012414.
2. fusiformis Methionin genus bacillus claimed in claim 1 is characterized in that tolerating 3% (w/v) urethanum.
3. the described fusiformis Methionin of claim 1 genus bacillus, it can synthesizing amino ethyl formate lytic enzyme.
4. the arbitrary described fusiformis Methionin genus bacillus of claim 1-3 is applied to the production of urethane ester hydrolase.
5. the arbitrary described fusiformis Methionin genus bacillus of claim 1-3 is applied to the degraded of urethanum.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103013948A (en) * | 2012-12-27 | 2013-04-03 | 江南大学 | Method for producing ethyl carbamate enzyme by purifying lysinibacillus fusiformis |
| CN103571765A (en) * | 2013-11-05 | 2014-02-12 | 江南大学 | Saccharomyces cerevisiae engineering bacteria with low-yielding ethyl carbamate, and building method and application of saccharomyces cerevisiae engineering bacteria |
| CN104232505A (en) * | 2014-07-14 | 2014-12-24 | 东华大学 | Lysinibacillus sp. DY2 bacterial strain and application thereof |
| WO2015035730A1 (en) * | 2013-09-10 | 2015-03-19 | 江南大学 | Ethyl carbamate hydrolase gene and protein encoded thereby and use thereof |
| CN105176886A (en) * | 2015-10-21 | 2015-12-23 | 湖南师范大学 | Lysinibacillus sphaericus and application of crystal protein and active product of Lysinibacillus sphaericus |
| CN106318893A (en) * | 2016-11-18 | 2017-01-11 | 江南大学 | Method for controlling ethyl carbamate in baijiu by means of lysinibacillus sphaericus |
| CN106566793A (en) * | 2016-11-14 | 2017-04-19 | 成都医学院 | Lysinibacillus sp. and application thereof in pesticide degrading |
-
2012
- 2012-12-27 CN CN201210578424.7A patent/CN103074275B/en active Active
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| J.V.WEBER ET.AL: "Ethyl carbamate in foods and beverages-a review", 《CLIMATE CHANGE,INTERCROPPING,PEST CONTROL AND BENEFICIAL MICROORGANISMS》 * |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103013948A (en) * | 2012-12-27 | 2013-04-03 | 江南大学 | Method for producing ethyl carbamate enzyme by purifying lysinibacillus fusiformis |
| CN103013948B (en) * | 2012-12-27 | 2015-04-15 | 江南大学 | Method for producing ethyl carbamate enzyme by purifying lysinibacillus fusiformis |
| WO2015035730A1 (en) * | 2013-09-10 | 2015-03-19 | 江南大学 | Ethyl carbamate hydrolase gene and protein encoded thereby and use thereof |
| CN103571765A (en) * | 2013-11-05 | 2014-02-12 | 江南大学 | Saccharomyces cerevisiae engineering bacteria with low-yielding ethyl carbamate, and building method and application of saccharomyces cerevisiae engineering bacteria |
| CN104232505A (en) * | 2014-07-14 | 2014-12-24 | 东华大学 | Lysinibacillus sp. DY2 bacterial strain and application thereof |
| CN105176886A (en) * | 2015-10-21 | 2015-12-23 | 湖南师范大学 | Lysinibacillus sphaericus and application of crystal protein and active product of Lysinibacillus sphaericus |
| CN105176886B (en) * | 2015-10-21 | 2019-04-16 | 湖南师范大学 | The application of one plant of spherical lysine bacillus and its crystalline protein and activated product |
| CN106566793A (en) * | 2016-11-14 | 2017-04-19 | 成都医学院 | Lysinibacillus sp. and application thereof in pesticide degrading |
| CN106318893A (en) * | 2016-11-18 | 2017-01-11 | 江南大学 | Method for controlling ethyl carbamate in baijiu by means of lysinibacillus sphaericus |
| CN106318893B (en) * | 2016-11-18 | 2019-08-20 | 江南大学 | A method of utilizing urethanes in lysine bacillus control white wine |
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