CN103074275B - 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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- CN103074275B CN103074275B CN201210578424.7A CN201210578424A CN103074275B CN 103074275 B CN103074275 B CN 103074275B CN 201210578424 A CN201210578424 A CN 201210578424A CN 103074275 B CN103074275 B CN 103074275B
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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 resistance to height of a kind of fusiformis Methionin genus bacillus and application thereof, particularly 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 fusiformis Methionin genus bacillus (Lysinibacillus fusiformis) SC02 of a strain product urethane ester hydrolase, 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 wax sample is opaque, and edge is irregular, colony diameter 2-4mm, and cultivating 1-2 days bacterium colonies is white, after 3 days, colony colour is deepened gradually, finally aobvious burgundy.Aerobic or amphimicrobian.Thalline Gram-positive, without pod membrane, under 1000 × microscope, 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: 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 dimorphism nutrient broth medium, 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 application of above-mentioned fusiformis Methionin genus bacillus, 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 40U/g that lives in the time of optimal pH 8.0, is greater than under 15% condition in NaCl concentration, and the resistance to height that fusiformis Methionin genus bacillus produces oozes urethane ester hydrolase and can keep 20% enzyme to live.
Fusiformis Methionin genus bacillus provided by the invention can be synthesized resistance to height and be oozed urethane ester hydrolase, is to have the highest enzyme at 8.0 o'clock to live at pH, can ooze fast decoupled urethanum under condition at height.Under the condition that is 8.0 at pH, its urethane ester hydrolase enzyme is lived as 40U/g, is greater than under 15% condition in NaCl concentration, and 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
The tolerance of Fig. 3 fusiformis Methionin genus bacillus urethane ester hydrolase to 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 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 under 1000 × g centrifugal 10 minutes, 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, cultivate after 24h in 37 ℃, 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.Carry out the extraction of enzyme extract by ultrasonication, crude enzyme liquid is carried out to urethane ester hydrolase enzyme activity determination and detect that a strain has the bacterial strain of urethanum hydrolytic enzyme activities, be a strain fusiformis Methionin genus bacillus by known this bacterial strain of 16S rDNA sequential analysis, be preserved in Chinese allusion quotation shape microbial strains preservation administrative center on October 19th, 2012, deposit number is CCTCC NO:M2012414, and 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 on nutrient broth agar solid medium, cultivate after 24h in 37 ℃, 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 phosphate buffered saline buffer 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 swellingly, makes cellularstructure fragmentation.By the bacterium liquid of multigelation, be placed in ice-water bath and 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 change of thalline solution is limpid, approximately 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 ultrapure water is settled to 250mL
Developer II: take 13.125g sodium hydroxide and 7.5mL clorox ultrapure water is settled to 250mL
Terminator: take 10g trichoroacetic acid(TCA) ultrapure water and be settled to 100mL
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, colorimetric estimation OD value under 630nm, and 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, respectively add 1mL trichoroacetic acid(TCA) terminator at two pipes, after mixing, add developer I and the 1mL developer II of 1mL, strong concussion, takes out after continuing to be incubated 20min in 37 ℃ of constant water bath box, is diluted to 10mL with ultrapure water, 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 (min) (time of enzyme reaction is 15min) of enzyme reaction
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: urethane ester hydrolase crude enzyme liquid optimal pH determine
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.Carrying out 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 definite urethane ester hydrolase.
Embodiment 5: the research of urethanum crude enzyme liquid salt tolerance
Enzyme liquid is respectively to 0%, 5% in NaCl concentration, 10%, in 15%, 18% damping fluid, under living stable lesser temps, enzyme is incubated 20min, the enzyme of measuring under corresponding conditions according to the method in embodiment tri-is lived, and draws the relation curve of salt concn-remnant enzyme activity and sees Fig. 3, determines the salt stability of enzyme.
Claims (5)
1. fusiformis Methionin genus bacillus (Lysinibacillus fusiformis) SC02 of urethane ester hydrolase is produced in a strain, is preserved in Chinese allusion quotation shape culture collection center on October 19th, 2012, and deposit number is CCTCC No:M2012414.
2. fusiformis Methionin genus bacillus claimed in claim 1, is characterized in that tolerating the urethanum that mass body integration number is 3%.
3. fusiformis Methionin genus bacillus described in claim 1, 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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| CN103013948B (en) * | 2012-12-27 | 2015-04-15 | 江南大学 | Method for producing ethyl carbamate enzyme by purifying lysinibacillus fusiformis |
| CN103451216B (en) * | 2013-09-10 | 2015-06-03 | 江南大学 | Ethyl carbamate hydrolase gene, protein coded thereby and application |
| 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 |
| 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 |
| CN106566793B (en) * | 2016-11-14 | 2019-11-29 | 成都医学院 | A kind of lysine bacillus and its application in degrading pesticide |
| 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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