CN103045514A - Proteus mirabilis for synthetizing acidic ethyl urethane hydrolytic enzyme and application thereof - Google Patents
Proteus mirabilis for synthetizing acidic ethyl urethane hydrolytic enzyme and application thereof Download PDFInfo
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- CN103045514A CN103045514A CN2012105784232A CN201210578423A CN103045514A CN 103045514 A CN103045514 A CN 103045514A CN 2012105784232 A CN2012105784232 A CN 2012105784232A CN 201210578423 A CN201210578423 A CN 201210578423A CN 103045514 A CN103045514 A CN 103045514A
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- proteus mirabilis
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- ethyl urethane
- urethanum
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Abstract
The invention discloses proteus mirabilis SW03 for synthetizing acidic ethyl urethane hydrolytic enzyme, which is preserved in China General Microbiological Culture Collection Center on October 19 th, 2012, and has the preservation number being CCTCCM2012416. The proteus mirabilis SW03 can rapidly degrade ethyl urethane and urea in the acidic environment, and plays a positive role in eliminating ethyl urethane in fermented food.
Description
Technical field
Screening and Identification and the slightly research of enzyme extract degraded urethanum characteristic thereof of the Proteus mirabilis of acid urethane ester hydrolase is produced in the present invention one strain Proteus mirabilis and application thereof, particularly a strain.
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.The research in recent years discovery, urethanum is a kind of human 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.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, realize the nothing harmization of urethanum in the food with microorganism and enzyme liberating, be comparatively ideal solution.
Proteus mirabilis (Proteus mirabilis) extensively is present in the enteron aisle of the organism of water, soil corruption and humans and animals.Generally not pathogenic in enteron aisle.Can rapid decomposing urea.This O antigen that belongs to bacterium has with some rickettsial incomplete antigen and intersects, and alternative rickettsial antigen and patients serum do agglutination reaction, and this reaction is called outer striking test, for the auxiliary diagnosis of some rickettsiosis.
Summary of the invention
Proteus mirabilis (Proteusmirabilis) SW03 that the purpose of this invention is to provide a strain synthetic acidic urethane ester hydrolase, be preserved in Chinese Typical Representative microbial strains preservation administrative center on October 19th, 2012, deposit number is CCTCC M2012416, and the preservation address is Wuhan, China Wuhan University.The morphological feature of thalline is that bacterium colony is circular, and surface drying is smooth, regular edges, incubation time is transparent less than 3 days bacterium colonies, it be yellow translucent cultivating bacterium colony greater than three days, the big or small 0.2-0.5mm of bacterium colony, available nutrient broth medium aerated culture or leave standstill cultivation with MRS substratum anaerobism.The thalline Gram-negative has pod membrane, and 1000 * microscopically observation of cell is rod-short, sees accompanying drawing 1.
Described Proteus mirabilis separates from the female mice stomach 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 two type nutrient broth mediums, 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 extracting method of above-mentioned Proteus mirabilis crude enzyme liquid.
Another object of the present invention provides the application of above-mentioned Proteus mirabilis, its synthetic acid urethane ester hydrolase can pH less than 5 condition under in the fast decoupled leavened food the by product urethanum and produce ethanol and ammonia.
Another object of the present invention provides the ureaclastic application under sour environment of above-mentioned Proteus mirabilis, its synthetic acid urease can pH less than 5 condition under in the fast decoupled leavened food by product urea and produce carbonic acid gas and ammonia with and the detection method of urease activity.
Proteus mirabilis energy synthesizing amino ethyl formate lytic enzyme provided by the invention and urase have the highest enzyme and live when pH4.8, can be at fast decoupled urethanum and urea under the acidic conditions.Be under 4.8 the condition at pH, its urethane ester hydrolase enzyme is lived and is that 11.7U/g, urase enzyme live and is 10.2U/g.
Figure of description
Fig. 1 Proteus mirabilis Proteus mirabilis Photomicrograph
The impact that Fig. 2 pH lives on Proteus mirabilis urethane ester hydrolase enzyme
Embodiment
Embodiment 1: the Proteus mirabilis 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 GI tract tissue with 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 the enrichment medium, urethanum 10g, water 1000mL, pH7.0), 30 ℃ of lower aerated culture 20h.Nutrient solution centrifugal 5 minutes in 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 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 activity of urethane ester hydrolase, by 16S rDNA sequential analysis as can be known this bacterial strain be a strain Proteus mirabilis, be preserved in Chinese Typical Representative microbial strains preservation administrative center on October 19th, 2012, deposit number is CCTCC M2012416, and the preservation address is Wuhan, China Wuhan University.。
Embodiment 2: the extracting method of urethane ester hydrolase crude enzyme liquid in the Proteus mirabilis
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 PBS damping fluid 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.Bacterium liquid with multigelation places ice-water bath to carry out ultrasonication, ultrasound condition: 25W, work 2S, interval 2S, repeated work 10min, until the thalline solution becomes limpid till, about spended time 20min.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 3.0% 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
6307 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 of analytic curve and definite urethane ester hydrolase.The collection of illustrative plates that drafting obtains is seen accompanying drawing 2.
Embodiment 5: the determining of Proteus mirabilis enzyme extract degraded urea ability
Solution preparation:
The DAM(Diacetylmonoxime) solution: accurately take by weighing 0.625g DAM, add the ultrapure water low-grade fever and stir, be settled to 25mL, place refrigerator stand-by
The TSC(thiosemicarbazide) solution accurately takes by weighing 0.0625g TSC, adds the ultrapure water low-grade fever and stirs, and is settled to 25mL, places refrigerator stand-by
Acid reagent: the strong phosphoric acid of 300mL85% and the vitriol oil of 10mL98% are mixed, be settled to 500mL
Developer: press DAM liquid: TSC liquid: sour reagent 2.5:1:50 mixes
Urea standardized solution preparation: accurately take by weighing 600mg urea and be dissolved in the 100mL water, be made into the urea soln of 0.1mol/L, and as mother liquor, with ultrapure water with it dilution and be mixed with the urea standardized solution of 0.1mmol/L, 0.2mmol/L, 0.4mmol/L, 0.6mmol/L, 0.8mmol/L, 1.0mmol/L.
Enzyme activity determination:
Get two test tubes, add respectively an amount of urea soln, wherein one is added thick zyme extract, simultaneously at 37 ℃ of lower reaction 15min.The 15mL developer is added in the 25mL colorimetric cylinder, and ultrapure water is settled to 25mL, stirs, and heats 27min in the boiling water bath, takes out and cool off 13min in tap water, colorimetric under 527nm.
Formula is calculated in enzyme work: enzyme activity=Δ OD
527* n * 10 * k/15
In the formula: Δ OD
527: the sample determination optical density value is poor after blank test and 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 activity definition: to decompose 1 μ mol urea be an enzyme activity unit to per minute under normal pressure, 37 ℃ of conditions.
Record under pH4.8, the enzyme of its urase is lived and is 10.2U/g.
Claims (5)
1. the Proteus mirabilis SW03 (Proteus mirabilisSW03) of a strain synthetic acidic urethane ester hydrolase is preserved in Chinese Typical Representative culture collection center on October 19th, 2012, and deposit number is CCTCC NO:M2012416.
2. Proteus mirabilis claimed in claim 1 is characterized in that tolerating 3% (w/v) urethanum.
3. Proteus mirabilis claimed in claim 1, it is characterized in that can synthesizing amino ethyl formate lytic enzyme and urase.
4. the arbitrary described Proteus mirabilis of claim 1-3 is applied to the production of urethane ester hydrolase.
5. the arbitrary described Proteus mirabilis of claim 1-3 is applied to the degraded of urethanum.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105603009A (en) * | 2016-02-15 | 2016-05-25 | 江南大学 | Microorganism converting method |
| CN105624228A (en) * | 2016-02-15 | 2016-06-01 | 江南大学 | Microbial conversion method |
| CN105969694A (en) * | 2016-06-20 | 2016-09-28 | 江南大学 | Providencia for synthesizing ethanol-resistant urethanase and urease |
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| CN101215567A (en) * | 2007-12-26 | 2008-07-09 | 中国人民解放军疾病预防控制所 | Nucleic acid identification sequence and detection method for singular proteus |
| CN101469326A (en) * | 2007-12-25 | 2009-07-01 | 天津生物芯片技术有限责任公司 | Nucleotide specific to ITS of Balcillus proteus mirabilis and use thereof |
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Patent Citations (2)
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| CN101469326A (en) * | 2007-12-25 | 2009-07-01 | 天津生物芯片技术有限责任公司 | Nucleotide specific to ITS of Balcillus proteus mirabilis and use thereof |
| CN101215567A (en) * | 2007-12-26 | 2008-07-09 | 中国人民解放军疾病预防控制所 | Nucleic acid identification sequence and detection method for singular proteus |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105603009A (en) * | 2016-02-15 | 2016-05-25 | 江南大学 | Microorganism converting method |
| CN105624228A (en) * | 2016-02-15 | 2016-06-01 | 江南大学 | Microbial conversion method |
| CN105624228B (en) * | 2016-02-15 | 2019-03-15 | 江南大学 | A kind of method of microorganism conversion |
| CN105603009B (en) * | 2016-02-15 | 2019-03-15 | 江南大学 | A kind of method of microorganism conversion |
| CN105969694A (en) * | 2016-06-20 | 2016-09-28 | 江南大学 | Providencia for synthesizing ethanol-resistant urethanase and urease |
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