CN104371987A - Method for separating yak stomach lysozyme - Google Patents
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- CN104371987A CN104371987A CN201410603940.XA CN201410603940A CN104371987A CN 104371987 A CN104371987 A CN 104371987A CN 201410603940 A CN201410603940 A CN 201410603940A CN 104371987 A CN104371987 A CN 104371987A
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Abstract
The invention discloses a method for separating and purifying yak stomach lysozyme. The method comprises the following steps: (1) homogenizing to obtain homogenate; (2) carrying out coarse purification to obtain a coarse purified solution; (3) carrying out CM-Sepharose FF column chromatography to obtain a chromatographic solution; (4) carrying out Sephadex G-75 column chromatography to obtain a yak stomach lysozyme solution; and (5) and lyophilizing to obtain yak stomach lysozyme. By using the method disclosed by the invention, the pure yak stomach lysozyme can be separated from yak gastric mucosal tissue, has high enzyme activity, wide application range and antibacterial effect and can be used as an antibacterial agent. Meanwhile, the method is high in yield and simple and convenient to operate and has industrial application values.
Description
Technical field
The present invention relates to a kind of separation method of yak stomach N,O-Diacetylmuramidase.
Background technology
N,O-Diacetylmuramidase is a kind of basic protein, and it is distributed widely in animal, plant and microorganism, expressed by all having in the histocytes such as mammiferous blood plasma, tracheae, enteron aisle, stomach, kidney and liver.N,O-Diacetylmuramidase, by destroying the β-Isosorbide-5-Nitrae glycosidic link in peptidoglycan between-acetylmuramic acid and NAG, causes cell wall structure to change, thus makes bacterolysis.N,O-Diacetylmuramidase owing to having germ resistance, and without toxicity, side effect, can be used as potential Substitutes For Antibiotic and applies to the fields such as food, biotechnology, medical treatment and animal and fowl fodder.From the viewpoint of disease of domestic animals control, N,O-Diacetylmuramidase is used in as additive in feed, effectively can prevent and treat scouring, simultaneously for it provides nutrition and enteron aisle protection, thus the death that the diarrhoea reducing domestic animal causes, improve its rate of animals delivered to the slaughter-house.
Yak, as the ruminating animal being distributed in extremely frigid zones, is the most important domestic animal in Qinghai-xizang Plateau Region.Due to the difference between itself and hinterland animal living enviroment, create the genetic specificity of yak self and the unique adaptability to environment.But the bibliographical information up to now, had no about separation and purification yak stomach N,O-Diacetylmuramidase.
Summary of the invention
In order to solve the problem, the invention provides a kind of separation purification method of yak abomasum N,O-Diacetylmuramidase.
The separation purification method of a kind of yak stomach of the present invention N,O-Diacetylmuramidase, comprises the steps:
(1) homogenate: get yak abomasum mucosa tissue, smash to pieces, add the Spirit of Mindererus of mucosal tissue 2 times (v/w), homogenate, centrifugal, separation of supernatant and precipitation, the Spirit of Mindererus of precipitation 2 times of weight (w/w) is added again again in precipitation, centrifugal, merge supernatant liquor, be homogenate; Wherein, the concentration of Spirit of Mindererus is 10mM;
(2) slightly pure: the pH to 4 regulating homogenate, centrifugal, get supernatant liquor, in boiling water bath, heat 2min, treat that temperature is down to room temperature, then adjust pH to 5, centrifugal, obtain supernatant, be thick Pure liquid;
(3) CM-Sepharose FF column chromatography: employing concentration is the Spirit of Mindererus balance CM-Sepharose FF post of 10mM, get the thick pure liquid loading of step (2), with the ammonium acetate wash-out foreign protein that concentration is 10mM, be the Spirit of Mindererus linear gradient elution of 10mM ~ 300mM again by concentration, according to wash-out
figurespectrum, collects the solution that second peak is corresponding, is chromatographic solution;
(4) Sephadex G-75 column chromatography: adopt concentration to be 0.2% (v/v) acetum balance Sephadex G-75 post, get the chromatographic solution loading of step (3), with the acetum wash-out that concentration is 0.2% (v/v), according to wash-out
figurespectrum, collects the solution that last peak is corresponding, obtains yak stomach lysozyme soln;
(5) freeze-drying, obtains yak stomach N,O-Diacetylmuramidase.
In step (1) and step (2), the centrifugal force of described centrifugal employing is 27000g, and centrifugation time is 15min.
In step (2), first time adjusts pH to adopt Glacial acetic acid, and second time adjusts pH to adopt ammoniacal liquor.
In step (3), the speed of wash-out is 1ml/min.
In step (3), during described linear gradient elution, the cumulative volume of elutriant is 20CV.
CV, represents column volume.
In step (4), the speed of wash-out is 0.2ml/min.
In step (4), first on chromatography column, drip the acetum that 1ml concentration is 0.2%, then wash-out.
Present invention also offers yak stomach N,O-Diacetylmuramidase prepared by preceding method, and prepare the purposes in fodder additives or antibacterials.
Adopt the inventive method can be separated from yak gastric mucosa tissue and obtain sterling yak abomasum N,O-Diacetylmuramidase, its enzyme work, up to 29,268U/mg, all has activity in wider pH scope and ionic strength range, there is good thermal stability, there is resistance to stomach en-, applied range, there is commercial application value, also there is bacteriostatic action, as Antibiogics usage, as additive in feed, can also prevent and treat scouring.Meanwhile, the inventive method yield is high, easy and simple to handle, and prospects for commercial application is good.
Accompanying drawing explanation
figure1 yak stomach lysozyme C M-Sepharose FF ion exchange chromatography;
figure2 yak stomach N,O-Diacetylmuramidase Sephadex G-75 gel-filtrations;
figure3 purified product SDS-PAGE electrophoresis
figure;
figure4 purified product fungistatic effects;
figure5pH is on the impact of yak stomach lysozyme activity;
figure6 ionic strengths are on the impact of enzymic activity;
figure7 ionic strengths and pH are on the impact of enzymic activity;
figure8 temperature are on the impact of enzymic activity;
figure9 yak stomach lysozyme activities over time.
embodiment
The embodiment of form by the following examples, is described in further detail foregoing of the present invention.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following embodiment.All technology realized based on foregoing of the present invention all belong to scope of the present invention.
Experiment material and reagent:
Experiment material
Meiwa yak abomasum
Reagent
Instrument
Embodiment 1 separation purification method of the present invention
1, acquired for materials
Meiwa yak abomasum is collected in slaughterhouse, bluish white river, Chengdu.After abomasum is taken out, with physiological saline, it is cleaned.Put into ice chest subsequently, take back laboratory, be stored in-20 DEG C.
2, separation and purification
2.1 prepare stomach mucous membrane homogenate
(1) yak abomasum is placed in 4 DEG C of environment to thaw.Then stomach mucous membrane is torn, take 7g yak abomasum stomach mucous membrane, and add 10mM ammonium acetate (pH=6) 14ml wherein.Finally carry out homogenized (all the other stomach mucous membranes are stored in-80 DEG C) with tissue mashing machine.
(2) homogenate is moved in the 50ml centrifuge tube after sterilizing, carry out at 4 DEG C centrifugal (27,000 × g, 15 minutes).Subsequently the supernatant liquor after centrifugal is transferred in new 50ml centrifuge tube and (is stored in 4 DEG C).
(3) 10mM ammonium acetate (pH=6) is joined again (10mM ammonium acetate: residue=2:1 in the test tube containing stomach mucous membrane residue, the proportioning of the two is volume mass ratio), centrifugal at 4 DEG C (27,000 × g, 15 minutes).Supernatant liquor is proceeded in new centrifuge tube again and (be stored in 4 DEG C).
The thick purifying of 2.2 sample
(1) in supernatant liquor, Glacial acetic acid is dripped, adjustment pH of mixed to 4.Then at 4 DEG C with 27, the centrifugation of 000 × g 15 minutes.Subsequently supernatant liquor is transferred in new centrifuge tube.
(2) supernatant liquor after centrifugal is put into 100 DEG C of water, water-bath 2 minutes.Taking out subsequently makes its temperature be down to room temperature.
(3) ammoniacal liquor is dropwise added in supernatant liquor, adjustment pH to 5.Then at 4 DEG C with 27, the centrifugation of 000 × g 15 minutes.Again supernatant liquor is transferred in new centrifuge tube, and is stored in 4 DEG C.
2.3CM-Sepharose FF column chromatography
(1) CM Sepherose FF is installed on Bio-Rad protein purification instrument, and with balance liquid (10mM ammonium acetate), prepacked column is balanced.
(2) with 0.20 μm of strainer, sample supernatant is filtered, and the 2ml supernatant liquor after filtering is added in chromatographic system.
(3) use balance liquid (the 10mM ammonium acetate of pH=5.8) with the flow velocity rinse-system of 1ml/min, wash-out foreign protein.
(4) in chromatographic system, add elutriant 10mM ammonium acetate, progressively be increased to 300mM ammonium acetate (pH=8), with the flow velocity of 1ml/min, linear gradient elution (gradient: 0-100% elution buffer is carried out to CM-Sepharose FF ion exchange column, 20CV), and according to wash-out
figurecollect the solution containing stomach N,O-Diacetylmuramidase, and be stored in 4 DEG C.
Wash-out
figureas
figureshown in 1:
Solution containing elution peak is collected, and activity identification is carried out to it.Result shows, target protein is present in collection tube corresponding to second peak.
Therefore, solution corresponding to second peak is collected when substantial sepn.
2.4Sephadex G-75 column chromatography
(1) take 5 ~ 6g gel dry powder, after adding the elutriant of 10 times of volumes, be placed in the water-bath water-bath 2 hours of 100 DEG C.Midway is manually slowly stirred.As found tiny gel pieces, after gel precipitation, removing supernatant, repeating several times until gel-free fragment (constantly add appropriate elutriant in water-bath process, prevent gel slurry evaporate to dryness) in gelating soln.
(2) chromatography column is vertically mounted on indoor, and injects the elutriant of 1/5 column volume to it, in order to get rid of the air at the bottom of chromatography column.Slowly gel slurry is added subsequently along glass rod.Post bottom outlet is opened after post to be installed completes, chromatography column is connected with Bio-Rad protein purification instrument simultaneously, (fill in post process with elutriant to its balance of spending the night again, whether the moment observes in chromatography column has gentle the bubbling out of tomography to show, if there is driving bubble at once out of or re-start dress post).
(3) 1ml lysozyme soln is slowly joined gel surface (avoid impacting gel as far as possible, cause the unfairness of gel surface, thus affect separating effect), open chromatography column outlet subsequently.Enter after colloid until lysozyme soln, slowly drip 1ml elutriant (0.2% acetic acid) in gel surface, then use the elutriant of 0.2ml/min flow velocity (0.2% acetic acid) to carry out wash-out to chromatography column.
(4) according to wash-out
figure, collect the solution of N,O-Diacetylmuramidase, and with Freeze Drying Equipment, frozen dried carried out to sample, obtain N,O-Diacetylmuramidase of the present invention (sample retention after process is in-20 DEG C).
The absorbancy of protein solution after separation and purification is measured, as
figureshown in 2.Solution containing elution peak is collected, and its activity is measured.Result shows, target protein is present in collection tube corresponding to last peak.
Therefore, collect solution corresponding to last peak when substantial sepn and be lysozyme soln of the present invention.
3, detect
3.1SDS-PAGE electrophoresis
(1) SDS-PAGE polyacrylamide gel preparation, is shown in
table 1:
table 1sDS-PAGE polyacrylamide gel
Table.1SDS-PAGE polyacrylamide gel
(2) draw 10 μ l enzyme solution in PCR pipe, add 2 μ l 5 × SDS-PAGE Loading buffer, and mixed solution is placed in 100 DEG C of water, water-bath 15 minutes.
(3) gel prepared is put into electrophoresis chamber, then in gel, add mixed solution.With 80v voltage by mixed solution electrophoresis to concentrated glue and separation gel line of delimitation place, then to adjust voltage be that 120v is until electrophoresis terminates.
(4) running gel immigration is filled in the vessel of coomassie brilliant blue staining liquid, then be placed in shaking table slowly concussion 4 hours (at dyeing course preservative film closed container mouth).
(5) discard dye liquor, with water by gel rinsing several, then add Xylene Brilliant Cyanine G destainer, be placed in the slow concussion of shaking table and spend the night, until protein band occurs (with preservative film closed container mouth in decolorization).
3.2 Enzyme activity assay
Use N,O-Diacetylmuramidase detection kit to measure the stomach lysozyme activity after separation and purification, step is as follows:
(1) bacterium liquid, standard protein solution and testing sample solution are placed in the pre-temperature more than 5 minutes of 37 DEG C of thermostat containers.
(2) spectrophotometric wavelength is adjusted to 530nm, and with distilled water, the transparence of UV detector is adjusted to 100%.
(3) mix joining in cuvette at 37 DEG C of pre-warm bacterium liquid (2ml) and standard protein solution (0.2ml), and record the transparence changing value in 2 minutes.
(4) join at 37 DEG C of pre-warm bacterium liquid (2ml) and testing sample solution (0.2ml) in cuvette, and repeat the measuring method in (3), obtain transparence changing value.
(5) testing sample activity (U/ml) is calculated according to calculation formula (testing sample activity (U/ml)=(treating test tube transparence changing value ÷ standard protein transparence changing value) × standard protein activity (200U/ml)).
3.3 enzyme solution concentration determinations
Use Bradford quantification of protein test kit to measure the stomach lysozyme concentration after separation and purification, step is as follows:
(1) 0,10,20,30,40,50,60 μ l bovine serum albumins reference liquid (1mg/ml) are joined in PCR pipe respectively, then add PBS and supply 150 μ l.
(2) reference liquid in (1) is joined in the cuvette containing the Xylene Brilliant Cyanine G dye liquor of 2.85ml and mix, and at room temperature place 5 to 10 minutes.
(3) with spectrophotometric determination 595nm place, the light absorption value of the bovine serum albumin reference liquid of different content, and record numerical value (not contain the absorbance value of bovine serum albumin reference liquid as blank).
(4) according to the light absorption value obtained and bovine serum albumin content, drawing standard curve.
(5) 80 μ l lysozyme solns are joined in PCR pipe, and supply 150 μ l with PBS.
(6) the N,O-Diacetylmuramidase mixing solutions in (5) is joined in the cuvette containing 2.85ml Xylene Brilliant Cyanine G dye liquor and mix, and at room temperature place 5 to 10 minutes.
(7) with spectrophotometric determination 595nm place, the light absorption value of N,O-Diacetylmuramidase mixing solutions, and record numerical value.
(8) according to typical curve, calculate the protein content under corresponding light absorption value, thus calculate the protein concentration of the lysozyme soln after separation and purification.
3.4 Resistence research
Utilize N,O-Diacetylmuramidase to have the characteristic of bacteriolyze effect to gram-positive microorganism, utilize streptococcus aureus to check the anti-microbial property of separation and purification gained N,O-Diacetylmuramidase, step is as follows:
(1) in the culture dish after sterilizing, add LB solid medium (antibiotic-free) about the 8ml after sterilizing, and be placed in horizontal plane, treat that it solidifies.
(2) get 0.5 ~ 1ml streptococcus aureus bacterium liquid, pour in remaining LB solid medium, shake up.
(3) in the culture dish containing LB solid medium, equidistant placement 3 Oxford cups (10mm × 7.8mm × 6.0mm).Pour the LB solid medium (8ml) containing streptococcus aureus bacterium liquid into culture dish subsequently, be positioned over horizontal plane, treat that it solidifies.
(4) in No. 1 and No. 2 Oxford cups, add the yak stomach N,O-Diacetylmuramidase after 3.6ug and 2.6 μ g separation and purification respectively, No. 3 is negative control (adding 100 μ l deionized waters).Put it into subsequently in 37 DEG C of constant incubators and cultivate 24 hours, observations.
3.4 peptide fingerprints
figurespectrum
Yak stomach N,O-Diacetylmuramidase after purifying carries out SDS-PAGE gel electrophoresis, intercept the gel of yak stomach N,O-Diacetylmuramidase electrophoretic band part, send Shanghai Inst. of Life Science, CAS, adopt electrophoresis mass spectrograph (Applied Biosystem, MALDI-TOF/TOF 4800 type), by Chinese Pharmacopoeia 2010 editions two annex Ⅸ J mass spectroscopies, use peptide fingerprint
figurespectral technology carries out identification of proteins.
4, measurement result
4.1SDS-PAGE electrophoresis
figurespectrum analysis
Yak stomach N,O-Diacetylmuramidase separation and purification of the present invention obtained carries out SDS-PAGE gel electrophoresis, result as
figure3.Find through electrophoresis detection, only show a protein band in gel, show that the sample that separation and purification obtains obtains purifying.By contrasting with standard protein, infer that yak abomasum N,O-Diacetylmuramidase molecular weight is about 15KDa.
Experimental result illustrates, the present invention is separated and obtains sterling yak abomasum N,O-Diacetylmuramidase.
4.2 yak abomasum lysozyme soln purification result
Carry out activity and concentration determination to the protein solution obtained after the separation and purification each time of yak stomach mucous membrane homogenate, its purification result is shown in
table 2.
table 2the separation and purification of yak stomach N,O-Diacetylmuramidase
upper tableshow, after separation and purification, the final yak stomach N,O-Diacetylmuramidase specific activity obtained is 29,268U/mg, purification is 66.51 times, the rate of recovery is 58.6%.
Known as calculated, about containing 1.99mg N,O-Diacetylmuramidase in 10g stomach mucous membrane.Meanwhile, adopt the inventive method, can extract 1.117mg N,O-Diacetylmuramidase from 10g stomach mucous membrane, yield is 58.6%.
Experimental result, the present invention is separated the sterling yak abomasum N,O-Diacetylmuramidase obtained, and enzyme is lived high.The inventive method yield is high simultaneously.
4.3 identification of proteins results
Shanghai Inst. of Life Science, CAS qualification result shows, this albumen is ox N,O-Diacetylmuramidase 2 precursor (lysozyme 2a precursor, partial, Bos taurus).Show the product of our separation and purification be a N,O-Diacetylmuramidase similar to ox N,O-Diacetylmuramidase (
figure10,11).
4.4 yak stomach N,O-Diacetylmuramidase fungistatic effects
Through qualification, the yak stomach N,O-Diacetylmuramidase of separation and purification gained to streptococcus aureus have obvious fungistatic effect (
figure4).Wherein, No. 1 inhibition zone is slightly large than No. 2 inhibition zones, and show the increase along with stomach N,O-Diacetylmuramidase consumption, its fungistatic effect also strengthens thereupon.
Experimental result illustrates, yak abomasum N,O-Diacetylmuramidase of the present invention has bacteriostatic activity, can be used for preparing antibacterials.
Experimental result illustrates, adopt the inventive method to be separated and obtain sterling yak abomasum N,O-Diacetylmuramidase, its enzyme is lived high, has bacteriostatic action.The yield of the inventive method is high simultaneously, is suitable for industrial application.
Embodiment 2 adopts the enzymatic property of the yak abomasum N,O-Diacetylmuramidase of the inventive method separation and purification
Get yak abomasum N,O-Diacetylmuramidase prepared by the embodiment of the present invention 1, detect its enzymatic property:
1, test method
1.1 buffer
Utilize the online calculation server of damping fluid
(http://www.biomol.net/en/tools/buffercalculator.htm) damping fluid to different pH and ionic strength calculates, and determines formula.
1.2pH is on the impact of enzymic activity
In room temperature environment, under being determined at same ion intensity (ionic strength=0.133), different pH is on the impact of enzymatic properties, and step is as follows:
(1) the buffer solution micrococcus luteus (ultimate density is 0.25mg/ml) of different pH is prepared, and 25 DEG C of insulations 1 hour.
(2) separation and purification gained lysozyme soln (with deionized water dissolving) is positioned over 37 DEG C of insulations 1 hour.
(3) draw 150 μ l lysozyme solns (5 μ g N,O-Diacetylmuramidase), join in 2ml micrococcus luteus solution, mixing of turning upside down.Then put into ultraviolet spectrophotometer (wavelength is 530nm) and measure transmittance at the changing value of 5 minutes, and record result.
(4) using N,O-Diacetylmuramidase at the transmittance changing value of pH=2 as 1, the transmittance changing value under all the other pH divided by the actual transmittance changing value of pH=2, in this, as the change multiple of the transmittance under this pH.
Different pH damping fluid:
1.10mM phosphoric acid buffer (PH=2, ionic strength=0.133): take 0.245g phosphoric acid and 1.8755g NaCl, is dissolved in 200ml deionized water, regulates pH to 2, is settled to 250ml.
2.10mM phosphoric acid buffer (PH=3, ionic strength=0.133): take 0.245g phosphoric acid and 1.814g NaCl, is dissolved in 200ml deionized water, regulates pH to 3, is settled to 250ml.
3.10mM acetate buffer solution (PH=4, ionic strength=0.133): take 0.15g acetic acid and 1.919gNaCl, is dissolved in 200ml deionized water, regulates pH to 4, is settled to 250ml.
4.10mM acetate buffer solution (PH=5, ionic strength=0.133): take 0.15g phosphoric acid and 1.8445g NaCl, is dissolved in 200ml deionized water, regulates pH to 5, is settled to 250ml.
5.10mM phosphoric acid buffer (PH=6.2, ionic strength=0.133): take 0.3g biphosphate and receive and 1.7445g NaCl, be dissolved in 200ml deionized water, regulate pH to 6.2, be settled to 250ml.
6.10mM phosphoric acid buffer (PH=7, ionic strength=0.133): take 0.3g biphosphate and receive and 1.6255g NaCl, be dissolved in 200ml deionized water, regulate pH to 7, be settled to 250ml.
7.10mM phosphoric acid buffer (PH=8, ionic strength=0.133): take 0.3g biphosphate and receive and 1.526g NaCl, be dissolved in 200ml deionized water, regulate pH to 8, be settled to 250ml.
8.10mM Tris damping fluid (PH=9, ionic strength=0.133): take 0.3025g Tris-base and 1.927g NaCl, be dissolved in 200ml deionized water, regulates pH to 9, is settled to 250ml.
1.3 ionic strengths are on the impact of enzymic activity
In room temperature environment, under being determined at identical pH (pH=5), different ionic strength is on the impact of enzymatic properties, and step is as follows:
(1) the buffer solution micrococcus luteus (ultimate density is 0.25mg/ml) of different ionic strength is prepared, and 25 DEG C of insulations 1 hour.
(2) separation and purification gained lysozyme soln (with deionized water dissolving) is positioned over 37 DEG C of insulations 1 hour.
(3) 150 μ l lysozyme solns (5 μ g N,O-Diacetylmuramidase) are drawn, join in 2ml micrococcus luteus solution, to turn upside down mixing, then put into ultraviolet spectrophotometer (wavelength is 530nm) and measure transmittance at the changing value of 5 minutes, and record result.
(4) be that the transmittance changing value of 0.007 is as 1 using N,O-Diacetylmuramidase in ionic strength, transmittance changing value under all the other ionic strengths is the actual transmittance changing value of 0.007 divided by ionic strength, in this, as the change multiple of the transmittance under this ionic strength.
The damping fluid of determination of activity under different ionic strength:
1.10mM acetate buffer solution (PH=5, ionic strength=7mM): take 0.15g acetic acid and 0.065g NaCl, be dissolved in 200ml deionized water, regulates PH to 5, is settled to 250ml.
2.10mM acetate buffer solution (PH=5, ionic strength=20mM): take 0.15g acetic acid and 0.195g NaCl, be dissolved in 200ml deionized water, regulates PH to 5, is settled to 250ml.
3.10mM acetate buffer solution (PH=5, ionic strength=50mM): take 0.15g acetic acid and 0.632g NaCl, be dissolved in 200ml deionized water, regulates PH to 5, is settled to 250ml.
4.10mM acetate buffer solution (PH=5, ionic strength=125mM): take 0.15g acetic acid and 1.7275g NaCl, be dissolved in 200ml deionized water, regulates PH to 5, is settled to 250ml.
5.10mM acetate buffer solution (PH=5, ionic strength=200mM): take 0.15g acetic acid and 2.824g NaCl, be dissolved in 200ml deionized water, regulates PH to 5, is settled to 250ml.
6.10mM acetate buffer solution (PH=5, ionic strength=300mM): take 0.15g acetic acid and 4.287g NaCl, be dissolved in 200ml deionized water, regulates PH to 5, is settled to 250ml.
7.10mM acetate buffer solution (PH=5, ionic strength=500mM): take 0.15g acetic acid and 7.2135g NaCl, be dissolved in 200ml deionized water, regulates PH to 5, is settled to 250ml.
The impact of 1.4 temperature on enzymic activity and the thermostability of enzyme
Lysozyme soln is placed in respectively 4 DEG C, 25 DEG C, 37 DEG C, 45 DEG C, 50 DEG C, 60 DEG C, 80 DEG C and 100 DEG C of water-baths.Water-bath 15 minutes.Then 150 μ l lysozyme solns (5 μ g N,O-Diacetylmuramidase) are drawn, join (25 DEG C are incubated 1 hour) in 2ml micrococcus luteus solution, the change of transmittance in 5 minutes is measured by ultraviolet spectrophotometer (wavelength is 530nm), and using N,O-Diacetylmuramidase at the transmittance changing value of 4 DEG C as 1, transmittance changing value at all the other temperature divided by the actual transmittance changing value of 4 DEG C, in this, as the change multiple of the transmittance at this temperature.
The damping fluid of determination of activity under differing temps: 10mM phosphoric acid buffer (PH=6.2, ionic strength=50mM): take 0.3g biphosphate and receive and 1.7445g NaCl, be dissolved in 200ml deionized water, regulate PH to 6, be settled to 250ml.
For studying the thermostability of albumen, measuring the transmittance change of yak stomach N,O-Diacetylmuramidase at relative optimum temperuture and 100 DEG C, and calculating the retention of enzymic activity in 100 DEG C with this.
1.5 ionic strengths and pH are on the impact of enzymic activity
The buffer solution micrococcus luteus (ultimate density is 0.25 mg/ml) of preparation different ionic strength and pH, and be incubated at being placed in 25 DEG C.Then the lysozyme soln (5 μ g N,O-Diacetylmuramidase) of 150 μ l after 37 DEG C of insulations is drawn, join mixing in 2ml micrococcus luteus solution (25 DEG C are incubated 1 hour), measure the change of transmittance in 5 minutes by ultraviolet spectrophotometer (wavelength is 530nm).And respectively using the transmittance changing value of the pH=4 of N,O-Diacetylmuramidase in different ionic strength as 1, in all the other different ionic strength, the transmittance changing value of different pH is divided by the actual transmittance changing value of pH=4 in this ionic strength, in this, as the change multiple of the transmittance under this ionic strength and pH.
The damping fluid of determination of activity under different ionic strength and pH:
1.10mM acetate buffer solution (PH=4, ionic strength=20mM): take 0.15g acetic acid and 0.268g NaCl, be dissolved in 200ml deionized water, regulates PH to 4, is settled to 250ml.
2.10Mm phosphoric acid buffer (PH=6.2, ionic strength=20mM): take 0.3g SODIUM PHOSPHATE, MONOBASIC and 0.1075g NaCl, be dissolved in 200ml deionized water, regulates PH to 6.2, is settled to 250ml.
3.10Mm phosphoric acid buffer (PH=7, ionic strength=20mM): take 0.3g SODIUM PHOSPHATE, MONOBASIC and 0.0025g NaCl, be dissolved in 200ml deionized water, regulates PH to 7, is settled to 250ml.
4.10mM Tris damping fluid (PH=8, ionic strength=20mM): take 0.3g Tris-base and 0.209g NaCl, be dissolved in 200ml deionized water, regulates PH to 8, is settled to 250ml.
5.10mM acetate buffer solution (PH=4, ionic strength=50mM): take 0.15g acetic acid and 0.706g NaCl, be dissolved in 200ml deionized water, regulates PH to 4, is settled to 250ml.
6.10Mm phosphoric acid buffer (PH=6.2, ionic strength=50mM): take 0.3g SODIUM PHOSPHATE, MONOBASIC and 0.5395g NaCl, be dissolved in 200ml deionized water, regulates PH to 6.2, is settled to 250ml.
7.10Mm phosphoric acid buffer (PH=7, ionic strength=50mM): take 0.3g SODIUM PHOSPHATE, MONOBASIC and 0.428g NaCl, be dissolved in 200ml deionized water, regulates PH to 7, is settled to 250ml.
8.10Mm phosphoric acid buffer (PH=8, ionic strength=50mM): take 0.3g SODIUM PHOSPHATE, MONOBASIC and 0.3155g NaCl, be dissolved in 200ml deionized water, regulates PH to 8, is settled to 250ml.
9.10mM acetate buffer solution (PH=4, ionic strength=133mM): take 0.15g acetic acid and 1.919g NaCl, be dissolved in 200ml deionized water, regulates PH to 4, is settled to 250ml.
10.10mM phosphoric acid buffer (PH=6.2, ionic strength=133mM): take 0.3g biphosphate and receive and 1.7445g NaCl, be dissolved in 200ml deionized water, regulate PH to 6, be settled to 250ml.
11.10mM phosphoric acid buffer (PH=7, ionic strength=133mM): take 0.3g biphosphate and receive and 1.6255g NaCl, be dissolved in 200ml deionized water, regulate PH to 7, be settled to 250ml.
12.10mM phosphoric acid buffer (PH=8, ionic strength=133mM): take 0.3g biphosphate and receive and 1.526g NaCl, be dissolved in 200ml deionized water, regulate PH to 8, be settled to 250ml.
13.10mM acetate buffer solution (PH=4, ionic strength=200mM): take 0.15g acetic acid and 2.899g NaCl, be dissolved in 200ml deionized water, regulates PH to 4, is settled to 250ml.
14.10Mm phosphoric acid buffer (PH=6.2, ionic strength=200mM): take 0.3g SODIUM PHOSPHATE, MONOBASIC and 2.7209g NaCl, be dissolved in 200ml deionized water, regulates PH to 6.2, is settled to 250ml.
15.10Mm phosphoric acid buffer (PH=7, ionic strength=200mM): take 0.3g SODIUM PHOSPHATE, MONOBASIC and 2.599g NaCl, be dissolved in 200ml deionized water, regulates PH to 7, is settled to 250ml.
16.10Mm phosphoric acid buffer (PH=8, ionic strength=200mM): take 0.3g SODIUM PHOSPHATE, MONOBASIC and 2.505g NaCl, be dissolved in 200ml deionized water, regulates PH to 8, is settled to 250ml.
1.6 unconventional kinetic measurement
The buffer solution micrococcus luteus (ultimate density is 0.25mg/ml) of configuration pH=5, ionic strength=0.05, and be placed in 25 DEG C of insulations 1 hour.Then draw the lysozyme soln (5 μ g N,O-Diacetylmuramidase) of 150 μ l after 37 DEG C of insulations, join in 2ml micrococcus luteus solution and mix.The transmittance values (every 30 seconds records once) of different time is measured by ultraviolet spectrophotometer (wavelength is 530nm).
Unconventional kinetic measurement damping fluid:
1.10mM acetate buffer solution (PH=5, ionic strength=50mM): take 0.15g acetic acid and 0.632g NaCl, be dissolved in 200ml deionized water, regulates PH to 5, is settled to 250ml.
2.0.066M sodium phosphate: take 1.7g sodium phosphate, is dissolved in 80ml deionized water, is settled to 100ml.
1.7 stomach en-is on the impact of enzymic activity
(1) 0.25mg yak stomach N,O-Diacetylmuramidase being dissolved in 50ml pH is in the 0.01M HCl (NaCl containing 0.1M) of 2.
(2) 6mg stomach en-is dissolved in 1ml 0.066M sodium phosphate (pH=6.2).
(3) prepare the damping fluid (attached 1 reagent configuration) that pH is 5, ionic strength is 0.133 and dissolve micrococcus luteus (ultimate density is 0.25mg/ml), and 25 DEG C of insulations 1 hour.
(4) draw 9 μ l pepsin solutions and join (stomach N,O-Diacetylmuramidase: stomach en-=1:1) in 10ml lysozyme soln, and adjust pH to 2.75.
(5) experimental group adding stomach en-(pH=2.75) and the control group that do not add stomach en-(pH=2.75) are placed in 37 DEG C simultaneously hatch 1 hour, after taking-up, all adjust pH to 6.5 with 1M NaOH.
(6) N,O-Diacetylmuramidase (experimental group and control group) of 150 μ l after 37 DEG C of insulations is drawn, join in 2ml micrococcus luteus solution, detect mixing solutions at 5 minutes iuuminting rate changing values by ultraviolet spectrophotometer (wavelength is 530nm).
2, test-results
2.1pH is on the impact of enzymic activity
In the buffered soln (ionic strength is 0.133) of different pH, measure stomach lysozyme activity, result as
figure5.
figurein visible, stomach N,O-Diacetylmuramidase of the present invention all has activity in the scope of pH2 ~ 9, wherein, is that in the weak acid environment of 5, activity is the highest at pH.
2.2 ionic strengths are on the impact of enzymic activity
In the buffered soln (pH is 5) of different ionic strength, measure the activity of stomach N,O-Diacetylmuramidase, result as
figure6.
From
figurein visible, stomach N,O-Diacetylmuramidase of the present invention all has activity in the scope that ionic strength is 0.007 ~ 0.3, and wherein, in the damping fluid of ionic strength=0.05, the activity of stomach N,O-Diacetylmuramidase is the highest.
Meanwhile, hypersaline environment (ionic strength > 0.05) the suppression degree to yak stomach lysozyme activity is better than low-salt environment (ionic strength < 0.05).
2.3 ionic strengths and pH are on the impact of enzymic activity
In the buffered soln of different pH value and ionic strength, measure the activity of stomach N,O-Diacetylmuramidase, result as
figure7.Laterally observe discovery: along with the increase (0.02 ~ 0.2) of ionic strength, the optimal pH of yak stomach N,O-Diacetylmuramidase is progressively moved to the meta-acid value that pH is 5 by the neutral value close to 7, therefore different ionic strengths can affect the optimum pH of yak stomach N,O-Diacetylmuramidase; Longitudinal 2 observation finds: along with the increase (0.02 ~ 0.2) of ionic strength, yak stomach lysozyme activity can decline (under optimal pH condition), namely in hypersaline environment, (ionic strength=0.2) yak stomach lysozyme activity can be suppressed, and compared with low-salt environment (ionic strength=0.02), stomach N,O-Diacetylmuramidase declines about 4 times at the antibacterial effect at optimal pH place.
The impact of 2.4 temperature on enzymic activity and the thermostability of enzyme
In differing temps environment, measure yak stomach lysozyme activity, result as
figure8.
figurein show: stomach N,O-Diacetylmuramidase of the present invention all has activity in the scope that temperature is 4 ~ 100 DEG C, and wherein, when temperature is 50 DEG C, its activity reaches the highest.
Experimental result illustrates, the relative optimum temperuture of yak stomach N,O-Diacetylmuramidase is at about 50 DEG C, and it is insensitive to temperature variation.
At high temperature measure the temperature stability of yak stomach N,O-Diacetylmuramidase, its result shows: yak stomach N,O-Diacetylmuramidase is 100 DEG C of water-baths after 15 minutes, and its activity still remains with this enzyme under optimum temperuture active about 73%.As can be seen here.Yak stomach N,O-Diacetylmuramidase has good thermostability in high temperature.
2.5 unconventional kinetics
Measure the time dependent rule of activity of stomach N,O-Diacetylmuramidase in the 10mM acetate buffer solution (pH=5, ionic strength=0.05), result as
figure9.By
figurein visible, along with the prolongation of time, the activity of yak stomach N,O-Diacetylmuramidase presents the trend that logarithm increases.
2.6 stomach en-s are on the impact of enzymic activity
Add the stomach en-of equivalent in yak stomach N,O-Diacetylmuramidase after, adjustment pH value of solution to 2.75, and hatch 1 hour at 37 DEG C.Use 1M NaOH that enzyme liquid pH is adjusted to 6.5 subsequently, and measure its activity.Result shows, through the gastric pepsin digestion stomach N,O-Diacetylmuramidase (experimental group) of 1 hour, its activity still remains with about 70% of original stomach N,O-Diacetylmuramidase (control group) activity.
As can be seen here, yak stomach N,O-Diacetylmuramidase is to pepsic strong resistance.
Experimental result illustrates, the sterling yak abomasum N,O-Diacetylmuramidase that the inventive method obtains has and all have activity in wider pH scope, ionic strength range and temperature range, have good thermostability, to stomach en-strong resistance, applied range.
To sum up, adopt separation method of the present invention can obtain sterling yak abomasum N,O-Diacetylmuramidase, its enzyme is lived high, in wider pH scope, ionic strength range and temperature range, all there is activity, have good thermostability, to pepsic strong resistance, also there is bacteriostatic action, as Antibiogics usage, or as additive in feed, scouring can be prevented and treated.Meanwhile, the inventive method yield is high, easy and simple to handle, has industrial application value.
Claims (10)
1. a separation purification method for yak stomach N,O-Diacetylmuramidase, is characterized in that: comprise the steps:
(1) homogenate: get yak abomasum mucosa tissue, smash to pieces, add Spirit of Mindererus, the add-on of Spirit of Mindererus is 2 times (v/w) of mucosal tissue, homogenate, centrifugal, separation of supernatant and precipitation, then Spirit of Mindererus is added again in precipitation, the add-on of Spirit of Mindererus is 2 times (v/w) of precipitation capacity, centrifugal, merge supernatant liquor, be homogenate; Wherein, the concentration of Spirit of Mindererus is 10mM;
(2) slightly pure: the pH to 4 regulating homogenate, centrifugal, get supernatant liquor, in boiling water bath, heat 2min, treat that temperature is down to room temperature, then adjust pH to 5, centrifugal, obtain supernatant, be thick pure liquid;
(3) CM-Sepharose FF column chromatography: employing concentration is the Spirit of Mindererus balance CM-Sepharose FF post of 10mM, get the thick pure liquid loading of step (2), with the ammonium acetate wash-out foreign protein that concentration is 10mM, be the Spirit of Mindererus linear gradient elution of 10mM ~ 300mM again by concentration, according to wash-out collection of illustrative plates, collect the solution that second peak is corresponding, be chromatographic solution;
(4) Sephadex G-75 column chromatography: adopt concentration to be 0.2% (v/v) acetum balance Sephadex G-75 post, get the chromatographic solution loading of step (3), with the acetum wash-out that concentration is 0.2% (v/v), according to wash-out collection of illustrative plates, collect the solution that last peak is corresponding, obtain yak stomach lysozyme soln;
(5) freeze-drying, obtains yak stomach N,O-Diacetylmuramidase.
2. separation purification method according to claim 1, is characterized in that: in step (1) and step (2), the centrifugal force of centrifugal employing is 27000g, and centrifugation time is 15min.
3. separation purification method according to claim 1, is characterized in that: in step (2), and first time adjusts pH to adopt Glacial acetic acid, and second time adjusts pH to adopt ammoniacal liquor.
4. separation purification method according to claim 1, is characterized in that: in step (3), and the speed of wash-out is 1ml/min.
5. separation purification method according to claim 1, is characterized in that: in step (3), during linear gradient elution, and the cumulative volume of elutriant is 20CV.
6. separation purification method according to claim 1, is characterized in that: in step (4), and the speed of wash-out is 0.2ml/min.
7. separation purification method according to claim 1, is characterized in that: in step (4), first on chromatography column, drips the acetum that 1ml concentration is 0.2%, then wash-out.
8. the yak stomach N,O-Diacetylmuramidase prepared of claim 1 ~ 7 any one method.
9. described in claim 8, the purposes in fodder additives prepared by yak stomach N,O-Diacetylmuramidase.
10. the purposes of yak stomach N,O-Diacetylmuramidase described in claim 8 in preparation antibacterials.
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| CN101352673A (en) * | 2007-07-26 | 2009-01-28 | 复旦大学 | Affinity chromatography for separating lysozyme and preparation method thereof |
| CN103160482A (en) * | 2013-03-14 | 2013-06-19 | 江苏大学 | Method for preparing egg white lysozyme and active protein by adopting coseparation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101352673A (en) * | 2007-07-26 | 2009-01-28 | 复旦大学 | Affinity chromatography for separating lysozyme and preparation method thereof |
| CN103160482A (en) * | 2013-03-14 | 2013-06-19 | 江苏大学 | Method for preparing egg white lysozyme and active protein by adopting coseparation |
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| MINGFENG JIANG ET AL.: "Yak (Bos Grunniens) Stomach Lysozyme: Molecular Cloning, Expression and its Antibacterial Activities", 《ANIMAL BIOTECHNOLOGY》 * |
| 任洪辉等: "牦牛胃溶菌酶基因在毕赤酵母中的分泌表达及活性分析", 《中国遗传学会第九次全国会员代表大会暨学术研讨会论文摘要汇编（2009-2013）》 * |
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